Microfluidic device with fault-tolerant multiple valve assembly

ABSTRACT

A microfluidic device having an inlet for receiving a liquid flowing through the microfluidic device, an outlet downstream of the inlet, and, a fault-tolerant multiple valve assembly having a plurality of flow-paths extending from the inlet to the outlet and a plurality of valves positioned along each of the flow-paths respectively.

FIELD OF THE INVENTION

The present invention relates to diagnostic devices that usemicrosystems technologies (MST). In particular, the invention relates tomicrofluidic and biochemical processing and analysis for moleculardiagnostics.

CO-PENDING APPLICATIONS

The following applications have been filed by the Applicant which relateto the present application:

GBS001US GBS002US GBS003US GBS005US GBS006US GSR001US GSR002US GAS001USGAS002US GAS003US GAS004US GAS006US GAS007US GAS008US GAS009US GAS010USGAS012US GAS013US GAS014US GAS015US GAS016US GAS017US GAS018US GAS019USGAS020US GAS021US GAS022US GAS023US GAS024US GAS025US GAS026US GAS027USGAS028US GAS030US GAS031US GAS032US GAS033US GAS034US GAS035US GAS036USGAS037US GAS038US GAS039US GAS040US GAS041US GAS042US GAS043US GAS044USGAS045US GAS046US GAS047US GAS048US GAS049US GAS050US GAS054US GAS055USGAS056US GAS057US GAS058US GAS059US GAS060US GAS061US GAS062US GAS063USGAS065US GAS066US GAS067US GAS068US GAS069US GAS070US GAS080US GAS081USGAS082US GAS083US GAS084US GAS085US GAS086US GAS087US GAS088US GAS089USGAS090US GAS091US GAS092US GAS093US GAS094US GAS095US GAS096US GAS097USGAS098US GAS099US GAS100US GAS101US GAS102US GAS103US GAS104US GAS105USGAS106US GAS108US GAS109US GAS110US GAS111US GAS112US GAS113US GAS114USGAS115US GAS117US GAS118US GAS119US GAS120US GAS121US GAS122US GAS123USGAS124US GAS125US GAS126US GAS127US GAS128US GAS129US GAS130US GAS131USGAS132US GAS133US GAS134US GAS135US GAS136US GAS137US GAS138US GAS139USGAS140US GAS141US GAS142US GAS143US GAS144US GAS146US GAS147US GRR001USGRR002US GRR003US GRR004US GRR005US GRR006US GRR007US GRR008US GRR009USGRR010US GVA001US GVA002US GVA005US GVA006US GVA007US GVA008US GVA009USGVA010US GVA011US GVA012US GVA013US GVA014US GVA015US GVA016US GVA017USGVA018US GVA019US GVA020US GVA021US GVA022US GHU001US GHU002US GHU003USGHU004US GHU006US GHU007US GHU008US GWM001US GWM002US GDI001US GDI002USGDI003US GDI004US GDI005US GDI006US GDI007US GDI009US GDI010US GDI011USGDI013US GDI014US GDI015US GDI016US GDI017US GDI019US GDI023US GDI028USGDI030US GDI039US GDI040US GDI041US GPC001US GPC002US GPC003US GPC004USGPC005US GPC006US GPC007US GPC008US GPC009US GPC010US GPC011US GPC012USGPC014US GPC017US GPC018US GPC019US GPC023US GPC027US GPC028US GPC029USGPC030US GPC031US GPC033US GPC034US GPC035US GPC036US GPC037US GPC038USGPC039US GPC040US GPC041US GPC042US GPC043US GLY001US GLY002US GLY003USGLY004US GLY005US GLY006US GIN001US GIN002US GIN003US GIN004US GIN005USGIN006US GIN007US GIN008US GMI001US GMI002US GMI005US GMI008US GLE001USGLE002US GLE003US GLE004US GLE005US GLE006US GLE007US GLE008US GLE009USGLE010US GLE011US GLE012US GLE013US GLE014US GLA001US GGA001US GGA003USGRE001US GRE002US GRE003US GRE004US GRE005US GRE006US GRE007US GCF001USGCF002US GCF003US GCF004US GCF005US GCF006US GCF007US GCF008US GCF009USGCF010US GCF011US GCF012US GCF013US GCF014US GCF015US GCF016US GCF020USGCF021US GCF022US GCF023US GCF024US GCF025US GCF027US GCF028US GCF029USGCF030US GCF031US GCF032US GCF033US GCF034US GCF035US GCF036US GCF037USGSA001US GSA002US GSE001US GSE002US GSE003US GSE004US GDA001US GDA002USGDA003US GDA004US GDA005US GDA006US GDA007US GPK001US GMO001US GMV001USGMV002US GMV003US GMV004US GRD001US GRD002US GRD003US GRD004US GPD001USGPD003US GPD004US GPD005US GPD006US GPD007US GPD008US GPD009US GPD010USGPD011US GPD012US GPD013US GPD014US GPD015US GPD016US GPD017US GAL001USGPA001US GPA003US GPA004US GPA005US GSS001US GSL001US GCA001US GCA002USGCA003US

The disclosures of these co-pending applications are incorporated hereinby reference. The above applications have been identified by theirfiling docket number, which will be substituted with the correspondingapplication number, once assigned.

BACKGROUND OF THE INVENTION

Molecular diagnostics has emerged as a field that offers the promise ofearly disease detection, potentially before symptoms have manifested.Molecular diagnostic testing is used to detect:

-   -   Inherited disorders    -   Acquired disorders    -   Infectious diseases    -   Genetic predisposition to health-related conditions.

With high accuracy and fast turnaround times, molecular diagnostic testshave the potential to reduce the occurrence of ineffective health careservices, enhance patient outcomes, improve disease management andindividualize patient care. Many of the techniques in moleculardiagnostics are based on the detection and identification of specificnucleic acids, both deoxyribonucleic acid (DNA) and ribonucleic acid(RNA), extracted and amplified from a biological specimen (such as bloodor saliva). The complementary nature of the nucleic acid bases allowsshort sequences of synthesized DNA (oligonucleotides) to bond(hybridize) to specific nucleic acid sequences for use in nucleic acidtests. If hybridization occurs, then the complementary sequence ispresent in the sample. This makes it possible, for example, to predictthe disease a person will contract in the future, determine the identityand virulence of an infectious pathogen, or determine the response aperson will have to a drug.

Nucleic Acid Based Molecular Diagnostic Test

A nucleic acid based test has four distinct steps:

1. Sample preparation

2. Nucleic acid extraction

3. Nucleic acid amplification (optional)

4. Detection

Many sample types are used for genetic analysis, such as blood, urine,sputum and tissue samples. The diagnostic test determines the type ofsample required as not all samples are representative of the diseaseprocess. These samples have a variety of constituents, but usually onlyone of these is of interest. For example, in blood, high concentrationsof erythrocytes can inhibit the detection of a pathogenic organism.Therefore a purification and/or concentration step at the beginning ofthe nucleic acid test is often required.

Blood is one of the more commonly sought sample types. It has threemajor constituents: leukocytes (white blood cells), erythrocytes (redblood cells) and thrombocytes (platelets). The thrombocytes facilitateclotting and remain active in vitro. To inhibit coagulation, thespecimen is mixed with an agent such as ethylenediaminetetraacetic acid(EDTA) prior to purification and concentration. Erythrocytes are usuallyremoved from the sample in order to concentrate the target cells. Inhumans, erythrocytes account for approximately 99% of the cellularmaterial but do not carry DNA as they have no nucleus. Furthermore,erythrocytes contain components such as haemoglobin that can interferewith the downstream nucleic acid amplification process (describedbelow). Removal of erythrocytes can be achieved by differentially lysingthe erythrocytes in a lysis solution, leaving remaining cellularmaterial intact which can then be separated from the sample usingcentrifugation. This provides a concentration of the target cells fromwhich the nucleic acids are extracted.

The exact protocol used to extract nucleic acids depends on the sampleand the diagnostic assay to be performed. For example, the protocol forextracting viral RNA will vary considerably from the protocol to extractgenomic DNA. However, extracting nucleic acids from target cells usuallyinvolves a cell lysis step followed by nucleic acid purification. Thecell lysis step disrupts the cell and nuclear membranes, releasing thegenetic material. This is often accomplished using a lysis detergent,such as sodium dodecyl sulfate, which also denatures the large amount ofproteins present in the cells.

The nucleic acids are then purified with an alcohol precipitation step,usually ice-cold ethanol or isopropanol, or via a solid phasepurification step, typically on a silica matrix in a column, resin or onparamagnetic beads in the presence of high concentrations of achaotropic salt, prior to washing and then elution in a low ionicstrength buffer. An optional step prior to nucleic acid precipitation isthe addition of a protease which digests the proteins in order tofurther purify the sample.

Other lysis methods include mechanical lysis via ultrasonic vibrationand thermal lysis where the sample is heated to 94° C. to disrupt cellmembranes.

The target DNA or RNA may be present in the extracted material in verysmall amounts, particularly if the target is of pathogenic origin.Nucleic acid amplification provides the ability to selectively amplify(that is, replicate) specific targets present in low concentrations todetectable levels.

The most commonly used nucleic acid amplification technique is thepolymerase chain reaction (PCR). PCR is well known in this field andcomprehensive description of this type of reaction is provided in E. vanPelt-Verkuil et al., Principles and Technical Aspects of PCRAmplification, Springer, 2008.

PCR is a powerful technique that amplifies a target DNA sequence againsta background of complex DNA. If RNA is to be amplified (by PCR), it mustbe first transcribed into cDNA (complementary DNA) using an enzymecalled reverse transcriptase. Afterwards, the resulting cDNA isamplified by PCR.

PCR is an exponential process that proceeds as long as the conditionsfor sustaining the reaction are acceptable. The components of thereaction are:

1. pair of primers—short single strands of DNA with around 10-30nucleotides complementary to the regions flanking the target sequence

2. DNA polymerase—a thermostable enzyme that synthesizes DNA

3. deoxyribonucleoside triphosphates (dNTPs)—provide the nucleotidesthat are incorporated into the newly synthesized DNA strand

4. buffer—to provide the optimal chemical environment for DNA synthesis

PCR typically involves placing these reactants in a small tube (˜10-50microlitres) containing the extracted nucleic acids. The tube is placedin a thermal cycler; an instrument that subjects the reaction to aseries of different temperatures for varying amounts of time. Thestandard protocol for each thermal cycle involves a denaturation phase,an annealing phase, and an extension phase. The extension phase issometimes referred to as the primer extension phase. In addition to suchthree-step protocols, two-step thermal protocols can be employed, inwhich the annealing and extension phases are combined. The denaturationphase typically involves raising the temperature of the reaction to90-95° C. to denature the DNA strands; in the annealing phase, thetemperature is lowered to ˜50-60° C. for the primers to anneal; and thenin the extension phase the temperature is raised to the optimal DNApolymerase activity temperature of 60-72° C. for primer extension. Thisprocess is repeated cyclically around 20-40 times, the end result beingthe creation of millions of copies of the target sequence between theprimers.

There are a number of variants to the standard PCR protocol such asmultiplex PCR, linker-primed PCR, direct PCR, tandem PCR, real-time PCRand reverse-transcriptase PCR, amongst others, which have been developedfor molecular diagnostics.

Multiplex PCR uses multiple primer sets within a single PCR mixture toproduce amplicons of varying sizes that are specific to different DNAsequences. By targeting multiple genes at once, additional informationmay be gained from a single test-run that otherwise would requireseveral experiments. Optimization of multiplex PCR is more difficultthough and requires selecting primers with similar annealingtemperatures, and amplicons with similar lengths and base composition toensure the amplification efficiency of each amplicon is equivalent.

Linker-primed PCR, also known as ligation adaptor PCR, is a method usedto enable nucleic acid amplification of essentially all DNA sequences ina complex DNA mixture without the need for target-specific primers. Themethod firstly involves digesting the target DNA population with asuitable restriction endonuclease (enzyme). Double-strandedoligonucleotide linkers (also called adaptors) with a suitableoverhanging end are then ligated to the ends of target DNA fragmentsusing a ligase enzyme. Nucleic acid amplification is subsequentlyperformed using oligonucleotide primers which are specific for thelinker sequences. In this way, all fragments of the DNA source which areflanked by linker oligonucleotides can be amplified.

Direct PCR describes a system whereby PCR is performed directly on asample without any, or with minimal, nucleic acid extraction. It haslong been accepted that PCR reactions are inhibited by the presence ofmany components of unpurified biological samples, such as the haemcomponent in blood. Traditionally, PCR has required extensivepurification of the target nucleic acid prior to preparation of thereaction mixture. With appropriate changes to the chemistry and sampleconcentration, however, it is possible to perform PCR with minimal DNApurification, or direct PCR. Adjustments to the PCR chemistry for directPCR include increased buffer strength, the use of polymerases which havehigh activity and processivity, and additives which chelate withpotential polymerase inhibitors.

Tandem PCR utilises two distinct rounds of nucleic acid amplification toincrease the probability that the correct amplicon is amplified. Oneform of tandem PCR is nested PCR in which two pairs of PCR primers areused to amplify a single locus in separate rounds of nucleic acidamplification. The first pair of primers hybridize to the nucleic acidsequence at regions external to the target nucleic acid sequence. Thesecond pair of primers (nested primers) used in the second round ofamplification bind within the first PCR product and produce a second PCRproduct containing the target nucleic acid, that will be shorter thanthe first one. The logic behind this strategy is that if the wrong locuswere amplified by mistake during the first round of nucleic acidamplification, the probability is very low that it would also beamplified a second time by a second pair of primers and thus ensuresspecificity.

Real-time PCR, or quantitative PCR, is used to measure the quantity of aPCR product in real time. By using a fluorophore-containing probe orfluorescent dyes along with a set of standards in the reaction, it ispossible to quantitate the starting amount of nucleic acid in thesample. This is particularly useful in molecular diagnostics wheretreatment options may differ depending on the pathogen load in thesample.

Reverse-transcriptase PCR (RT-PCR) is used to amplify DNA from RNA.Reverse transcriptase is an enzyme that reverse transcribes RNA intocomplementary DNA (cDNA), which is then amplified by PCR. RT-PCR iswidely used in expression profiling, to determine the expression of agene or to identify the sequence of an RNA transcript, includingtranscription start and termination sites. It is also used to amplifyRNA viruses such as human immunodeficiency virus or hepatitis C virus.

Isothermal amplification is another form of nucleic acid amplificationwhich does not rely on the thermal denaturation of the target DNA duringthe amplification reaction and hence does not require sophisticatedmachinery. Isothermal nucleic acid amplification methods can thereforebe carried out in primitive sites or operated easily outside of alaboratory environment. A number of isothermal nucleic acidamplification methods have been described, including Strand DisplacementAmplification, Transcription Mediated Amplification, Nucleic AcidSequence Based Amplification, Recombinase Polymerase Amplification,Rolling Circle Amplification, Ramification Amplification,Helicase-Dependent Isothermal DNA Amplification and Loop-MediatedIsothermal Amplification.

Isothermal nucleic acid amplification methods do not rely on thecontinuing heat denaturation of the template DNA to produce singlestranded molecules to serve as templates for further amplification, butinstead rely on alternative methods such as enzymatic nicking of DNAmolecules by specific restriction endonucleases, or the use of an enzymeto separate the DNA strands, at a constant temperature.

Strand Displacement Amplification (SDA) relies on the ability of certainrestriction enzymes to nick the unmodified strand of hemi-modified DNAand the ability of a 5′-3′ exonuclease-deficient polymerase to extendand displace the downstream strand. Exponential nucleic acidamplification is then achieved by coupling sense and antisense reactionsin which strand displacement from the sense reaction serves as atemplate for the antisense reaction. The use of nickase enzymes which donot cut DNA in the traditional manner but produce a nick on one of theDNA strands, such as N. Alw1, N. BstNB1 and Mly1, are useful in thisreaction. SDA has been improved by the use of a combination of aheat-stable restriction enzyme (Ava1) and heat-stable Exo-polymerase(Bst polymerase). This combination has been shown to increaseamplification efficiency of the reaction from 10⁸ fold amplification to10¹⁰ fold amplification so that it is possible using this technique toamplify unique single copy molecules.

Transcription Mediated Amplification (TMA) and Nucleic Acid SequenceBased Amplification (NASBA) use an RNA polymerase to copy RNA sequencesbut not corresponding genomic DNA. The technology uses two primers andtwo or three enzymes, RNA polymerase, reverse transcriptase andoptionally RNase H (if the reverse transcriptase does not have RNaseactivity). One primer contains a promoter sequence for RNA polymerase.In the first step of nucleic acid amplification, this primer hybridizesto the target ribosomal RNA (rRNA) at a defined site. Reversetranscriptase creates a DNA copy of the target rRNA by extension fromthe 3′ end of the promoter primer. The RNA in the resulting RNA:DNAduplex is degraded by the RNase activity of the reverse transcriptase ifpresent or the additional RNase H. Next, a second primer binds to theDNA copy. A new strand of DNA is synthesized from the end of this primerby reverse transcriptase, creating a double-stranded DNA molecule. RNApolymerase recognizes the promoter sequence in the DNA template andinitiates transcription. Each of the newly synthesized RNA ampliconsre-enters the process and serves as a template for a new round ofreplication.

In Recombinase Polymerase Amplification (RPA), the isothermalamplification of specific DNA fragments is achieved by the binding ofopposing oligonucleotide primers to template DNA and their extension bya DNA polymerase. Heat is not required to denature the double-strandedDNA (dsDNA) template. Instead, RPA employs recombinase-primer complexesto scan dsDNA and facilitate strand exchange at cognate sites. Theresulting structures are stabilised by single-stranded DNA bindingproteins interacting with the displaced template strand, thus preventingthe ejection of the primer by branch migration. Recombinase disassemblyleaves the 3′ end of the oligonucleotide accessible to a stranddisplacing DNA polymerase, such as the large fragment of Bacillussubtilis Pol I (Bsu), and primer extension ensues. Exponential nucleicacid amplification is accomplished by the cyclic repetition of thisprocess.

Helicase-dependent amplification (HDA) mimics the in vivo system in thatit uses a DNA helicase enzyme to generate single-stranded templates forprimer hybridization and subsequent primer extension by a DNApolymerase. In the first step of the HDA reaction, the helicase enzymetraverses along the target DNA, disrupting the hydrogen bonds linkingthe two strands which are then bound by single-stranded bindingproteins. Exposure of the single-stranded target region by the helicaseallows primers to anneal. The DNA polymerase then extends the 3′ ends ofeach primer using free deoxyribonucleoside triphosphates (dNTPs) toproduce two DNA replicates. The two replicated dsDNA strandsindependently enter the next cycle of HDA, resulting in exponentialnucleic acid amplification of the target sequence.

Other DNA-based isothermal techniques include Rolling CircleAmplification (RCA) in which a DNA polymerase extends a primercontinuously around a circular DNA template, generating a long DNAproduct that consists of many repeated copies of the circle. By the endof the reaction, the polymerase generates many thousands of copies ofthe circular template, with the chain of copies tethered to the originaltarget DNA. This allows for spatial resolution of target and rapidnucleic acid amplification of the signal. Up to 10¹² copies of templatecan be generated in 1 hour. Ramification amplification is a variation ofRCA and utilizes a closed circular probe (C-probe) or padlock probe anda DNA polymerase with a high processivity to exponentially amplify theC-probe under isothermal conditions.

Loop-mediated isothermal amplification (LAMP), offers high selectivityand employs a DNA polymerase and a set of four specially designedprimers that recognize a total of six distinct sequences on the targetDNA. An inner primer containing sequences of the sense and antisensestrands of the target DNA initiates LAMP. The following stranddisplacement DNA synthesis primed by an outer primer releases asingle-stranded DNA. This serves as template for DNA synthesis primed bythe second inner and outer primers that hybridize to the other end ofthe target, which produces a stem-loop DNA structure. In subsequent LAMPcycling one inner primer hybridizes to the loop on the product andinitiates displacement DNA synthesis, yielding the original stem-loopDNA and a new stem-loop DNA with a stem twice as long. The cyclingreaction continues with accumulation of 10⁹ copies of target in lessthan an hour. The final products are stem-loop DNAs with severalinverted repeats of the target and cauliflower-like structures withmultiple loops formed by annealing between alternately inverted repeatsof the target in the same strand.

After completion of the nucleic acid amplification, the amplifiedproduct must be analysed to determine whether the anticipated amplicon(the amplified quantity of target nucleic acids) was generated. Themethods of analyzing the product range from simply determining the sizeof the amplicon through gel electrophoresis, to identifying thenucleotide composition of the amplicon using DNA hybridization.

Gel electrophoresis is one of the simplest ways to check whether thenucleic acid amplification process generated the anticipated amplicon.Gel electrophoresis uses an electric field applied to a gel matrix toseparate DNA fragments. The negatively charged DNA fragments will movethrough the matrix at different rates, determined largely by their size.After the electrophoresis is complete, the fragments in the gel can bestained to make them visible. Ethidium bromide is a commonly used stainwhich fluoresces under UV light.

The size of the fragments is determined by comparison with a DNA sizemarker (a DNA ladder), which contains DNA fragments of known sizes, runon the gel alongside the amplicon. Because the oligonucleotide primersbind to specific sites flanking the target DNA, the size of theamplified product can be anticipated and detected as a band of knownsize on the gel. To be certain of the identity of the amplicon, or ifseveral amplicons have been generated, DNA probe hybridization to theamplicon is commonly employed.

DNA hybridization refers to the formation of double-stranded DNA bycomplementary base pairing. DNA hybridization for positiveidentification of a specific amplification product requires the use of aDNA probe around 20 nucleotides in length. If the probe has a sequencethat is complementary to the amplicon (target) DNA sequence,hybridization will occur under favourable conditions of temperature, pHand ionic concentration. If hybridization occurs, then the gene or DNAsequence of interest was present in the original sample.

Optical detection is the most common method to detect hybridization.Either the amplicons or the probes are labelled to emit light throughfluorescence or electrochemiluminescence. These processes differ in themeans of producing excited states of the light-producing moieties, butboth enable covalent labelling of nucleotide strands. Inelectrochemiluminescence (ECL), light is produced by luminophoremolecules or complexes upon stimulation with an electric current. Influorescence, it is illumination with excitation light which leads toemission.

Fluorescence is detected using an illumination source which providesexcitation light at a wavelength absorbed by the fluorescent molecule,and a detection unit. The detection unit comprises a photosensor (suchas a photomultiplier tube or charge-coupled device (CCD) array) todetect the emitted signal, and a mechanism (such as awavelength-selective filter) to prevent the excitation light from beingincluded in the photosensor output. The fluorescent molecules emitStokes-shifted light in response to the excitation light, and thisemitted light is collected by the detection unit. Stokes shift is thefrequency difference or wavelength difference between emitted light andabsorbed excitation light.

ECL emission is detected using a photosensor which is sensitive to theemission wavelength of the ECL species being employed. For example,transition metal-ligand complexes emit light at visible wavelengths, soconventional photodiodes and CCDs are employed as photosensors. Anadvantage of ECL is that, if ambient light is excluded, the ECL emissioncan be the only light present in the detection system, which improvessensitivity.

Microarrays allow for hundreds of thousands of DNA hybridizationexperiments to be performed simultaneously. Microarrays are powerfultools for molecular diagnostics with the potential to screen forthousands of genetic diseases or detect the presence of numerousinfectious pathogens in a single test. A microarray consists of manydifferent DNA probes immobilized as spots on a substrate. The target DNA(amplicon) is first labelled with a fluorescent or luminescent molecule(either during or after nucleic acid amplification) and then applied tothe array of probes. The microarray is incubated in a temperaturecontrolled, humid environment for a number of hours or days whilehybridization between the probe and amplicon takes place. Followingincubation, the microarray must be washed in a series of buffers toremove unbound strands. Once washed, the microarray surface is driedusing a stream of air (often nitrogen). The stringency of thehybridization and washes is critical. Insufficient stringency can resultin a high degree of nonspecific binding. Excessive stringency can leadto a failure of appropriate binding, which results in diminishedsensitivity. Hybridization is recognized by detecting light emissionfrom the labelled amplicons which have formed a hybrid withcomplementary probes.

Fluorescence from microarrays is detected using a microarray scannerwhich is generally a computer controlled inverted scanning fluorescenceconfocal microscope which typically uses a laser for excitation of thefluorescent dye and a photosensor (such as a photomultiplier tube orCCD) to detect the emitted signal. The fluorescent molecules emitStokes-shifted light (described above) which is collected by thedetection unit.

The emitted fluorescence must be collected, separated from theunabsorbed excitation wavelength, and transported to the detector. Inmicroarray scanners, a confocal arrangement is commonly used toeliminate out-of-focus information by means of a confocal pinholesituated at an image plane. This allows only the in-focus portion of thelight to be detected. Light from above and below the plane of focus ofthe object is prevented from entering the detector, thereby increasingthe signal to noise ratio. The detected fluorescent photons areconverted into electrical energy by the detector which is subsequentlyconverted to a digital signal. This digital signal translates to anumber representing the intensity of fluorescence from a given pixel.Each feature of the array is made up of one or more such pixels. Thefinal result of a scan is an image of the array surface. The exactsequence and position of every probe on the microarray is known, and sothe hybridized target sequences can be identified and analysedsimultaneously.

More information regarding fluorescent probes can be found at:http://www.premierbiosoft.com/tech_notes/FRET_probe.html andhttp://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Technical-Notes-and-Product-Highlights/Fluorescence-Resonance-Energy-Transfer-FRET.html

Point-of-Care Molecular Diagnostics

Despite the advantages that molecular diagnostic tests offer, the growthof this type of testing in the clinical laboratory has been slower thanexpected and remains a minor part of the practice of laboratorymedicine. This is primarily due to the complexity and costs associatedwith nucleic acid testing compared with tests based on methods notinvolving nucleic acids. The widespread adaptation of moleculardiagnostics testing to the clinical setting is intimately tied to thedevelopment of instrumentation that significantly reduces the cost,provides a rapid and automated assay from start (specimen processing) tofinish (generating a result) and operates without major intervention bypersonnel.

A point-of-care technology serving the physician's office, the hospitalbedside or even consumer-based, at home, would offer many advantagesincluding:

-   -   rapid availability of results enabling immediate facilitation of        treatment and improved quality of care.    -   ability to obtain laboratory values from testing very small        samples.    -   reduced clinical workload.    -   reduced laboratory workload and improved office efficiency by        reducing administrative work.    -   improved cost per patient through reduced length of stay of        hospitalization, conclusion of outpatient consultation at the        first visit, and reduced handling, storing and shipping of        specimens.    -   facilitation of clinical management decisions such as infection        control and antibiotic use.

Lab-on-a-Chip (LOC) Based Molecular Diagnostics

Molecular diagnostic systems based on microfluidic technologies providethe means to automate and speed up molecular diagnostic assays. Thequicker detection times are primarily due to the extremely low volumesinvolved, automation, and the low-overhead inbuilt cascading of thediagnostic process steps within a microfluidic device. Volumes in thenanoliter and microliter scale also reduce reagent consumption and cost.Lab-on-a-chip (LOC) devices are a common form of microfluidic device.LOC devices have MST structures within a MST layer for fluid processingintegrated onto a single supporting substrate (usually silicon).Fabrication using the VLSI (very large scale integrated) lithographictechniques of the semiconductor industry keeps the unit cost of each LOCdevice very low. However, controlling fluid flow through the LOC device,adding reagents, controlling reaction conditions and so on necessitatebulky external plumbing and electronics. Connecting a LOC device tothese external devices effectively restricts the use of LOC devices formolecular diagnostics to the laboratory setting. The cost of theexternal equipment and complexity of its operation precludes LOC-basedmolecular diagnostics as a practical option for point-of-care settings.

In view of the above, there is a need for a molecular diagnostic systembased on a LOC device for use at point-of-care.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a microfluidic devicecomprising:

an inlet for receiving a liquid flowing through the microfluidic device;

an outlet downstream of the inlet; and,

a fault-tolerant multiple valve assembly having a plurality offlow-paths extending from the inlet to the outlet and a plurality ofvalves positioned along each of the flow-paths respectively.

Preferably, each of the flow-paths is configured for capillary drivenflow of the liquid towards the outlet, and each of the valves areconfigured to arrest flow towards the outlet until opened.

Preferably, the microfluidic device also has a sensor in each of theflow-paths respectively, the sensor being responsive to contact with theliquid.

Preferably, the valves are bend actuated valves, each having a movablemember configured for movement between a quiescent position and anactuated position displaced from the quiescent position and, an apertureat least partially defined by the movable member, the apertureconfigured to arrest the capillary driven flow by pinning a meniscus atthe aperture wherein during use, displacement of the movable member tothe actuated position unpins the meniscus from the aperture such thatthe capillary driven flow towards the outlet resumes.

Preferably, the bend actuated valve has a resistive element for causingdifferential thermal expansion to move the movable member.

Preferably, the movable member is a cantilevered structure with thenozzle at the free end and the resistive element between the nozzle andthe fixed end.

Preferably, the valves are thermally actuated valves, each having anaperture for anchoring a meniscus to arrest the capillary drive flow,and a valve heater for heating the meniscus such that the meniscusunpins from the aperture such that the capillary driven flow towards theoutlet resumes.

Preferably, the valve heater extends about the periphery of theaperture.

Preferably, each of the flow-paths has an upstream valve and adownstream valve, the sensor being positioned between the upstream valveand the downstream valves.

Preferably, the microfluidic device also has two of the flow-paths.

Preferably, the microfluidic device also has CMOS circuitry connected tothe sensors for operative control of the valves and detecting faultsincluding failure of either of the upstream valves to arrest the liquidflow.

Preferably, the microfluidic device also has a polymerase chain reaction(PCR) section for amplifying nucleic acid sequences by thermally cyclingthe nucleic acid sequences and a PCR mix of reagents through adenaturation temperature, an annealing temperature and a primerextension temperature; wherein,

the fault-tolerant multiple valve assembly retains the nucleic acidsequences and a PCR mix of reagents in the PCR section during thethermal cycling.

Preferably, the microfluidic device also has an array of probes forhybridization with target nucleic acid sequences to form probe-targethybrids;

the array of probes being downstream of the fault-tolerant multiplevalve assembly such that opening the fault-tolerant multiple valveassembly allows amplicon from the PCR section to contact the probes.

Preferably, the PCR section is configured to produce sufficient ampliconfor hybridization with more than 1000 of the probes in less than 10minutes of thermal cycling.

Preferably, the PCR section has a thermal cycle time between 0.45seconds and 1.5 seconds.

Preferably, the microfluidic device also has a temperature sensor andwherein the PCR section has at least one heater for thermally cyclingthe nucleic acid sequences and the PCR mix, the temperature sensor andthe at least one heater being connected to the CMOS circuitry forfeedback control of the at least one heater.

Preferably, the CMOS circuitry opens the fault-tolerant valve assemblyafter a predetermined number of thermal cycles.

Preferably, the PCR section has a plurality of elongate PCR chambershaving a longitudinal extent much greater than their lateral dimensions,and a plurality of heaters, each of the heaters being elongate andparallel with the longitudinal extent of the PCR chambers.

Preferably, the plurality of elongate heaters are independentlyoperable.

Preferably, the microfluidic device also has an array of photodiodes fordetecting hybridization of probes within the array of probes.

The mass-producible and inexpensive microfluidic device accepts a liquidfor processing and/or analysis, with the requisite fluidic propulsionbeing provided via capillary action, and the requisite valvefunctionality being provided via reliable, easily manufacturablefault-tolerant multiple-valve assemblies.

Fault-tolerant multiple-valve assemblies provide the requisite level ofreliability via individual valve fault tolerance, and their design,fabrication, and operation exploit the intrinsic positive qualities ofmicrofluidic device technologies and avoid problematic aspects of suchtechnologies.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the present invention will now be described byway of example only with reference to the accompanying drawings, inwhich:

FIG. 1 shows a test module and test module reader configured forfluorescence detection;

FIG. 2 is a schematic overview of the electronic components in the testmodule configured for fluorescence detection;

FIG. 3 is a schematic overview of the electronic components in the testmodule reader;

FIG. 4 is a schematic representation of the architecture of the LOCdevice;

FIG. 5 is a perspective of the LOC device;

FIG. 6 is a plan view of the LOC device with features and structuresfrom all layers superimposed on each other;

FIG. 7 is a plan view of the LOC device with the structures of the capshown in isolation;

FIG. 8 is a top perspective of the cap with internal channels andreservoirs shown in dotted line;

FIG. 9 is an exploded top perspective of the cap with internal channelsand reservoirs shown in dotted line;

FIG. 10 is a bottom perspective of the cap showing the configuration ofthe top channels;

FIG. 11 is a plan view of the LOC device showing the structures of theCMOS+MST device in isolation;

FIG. 12 is a schematic section view of the LOC device at the sampleinlet;

FIG. 13 is an enlarged view of Inset AA shown in FIG. 6;

FIG. 14 is an enlarged view of Inset AB shown in FIG. 6;

FIG. 15 is an enlarged view of Inset AE shown in FIG. 13;

FIG. 16 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AE;

FIG. 17 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AE;

FIG. 18 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AE;

FIG. 19 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AE;

FIG. 20 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AE;

FIG. 21 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AE;

FIG. 22 is schematic section view of the lysis reagent reservoir shownin FIG. 21;

FIG. 23 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AB;

FIG. 24 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AB;

FIG. 25 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AI;

FIG. 26 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AB;

FIG. 27 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AB;

FIG. 28 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AB;

FIG. 29 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AB;

FIG. 30 is a schematic section view of the amplification mix reservoirand the polymerase reservoir;

FIG. 31 show the features of a boiling-initiated valve in isolation;

FIG. 32 is a schematic section view of the boiling-initiated valve takenthrough line 33-33 shown in FIG. 31;

FIG. 33 is an enlarged view of Inset AF shown in FIG. 15;

FIG. 34 is a schematic section view of the upstream end of the dialysissection taken through line 35-35 shown in FIG. 33;

FIG. 35 is an enlarged view of Inset AC shown in FIG. 6;

FIG. 36 is a further enlarged view within Inset AC showing theamplification section;

FIG. 37 is a further enlarged view within Inset AC showing theamplification section;

FIG. 38 is a further enlarged view within Inset AC showing theamplification section;

FIG. 39 is a further enlarged view within Inset AK shown in FIG. 38;

FIG. 40 is a further enlarged view within Inset AC showing theamplification chamber;

FIG. 41 is a further enlarged view within Inset AC showing theamplification section;

FIG. 42 is a further enlarged view within Inset AC showing theamplification chamber;

FIG. 43 is a further enlarged view within Inset AL shown in FIG. 42;

FIG. 44 is a further enlarged view within Inset AC showing theamplification section;

FIG. 45 is a further enlarged view within Inset AM shown in FIG. 44;

FIG. 46 is a further enlarged view within Inset AC showing theamplification chamber;

FIG. 47 is a further enlarged view within Inset AN shown in FIG. 46;

FIG. 48 is a further enlarged view within Inset AC showing theamplification chamber;

FIG. 49 is a further enlarged view within Inset AC showing theamplification chamber;

FIG. 50 is a further enlarged view within Inset AC showing theamplification section;

FIG. 51 is a schematic section view of the amplification section;

FIG. 52 is an enlarged plan view of the hybridization section;

FIG. 53 is a further enlarged plan view of two hybridization chambers inisolation;

FIG. 54 is schematic section view of a single hybridization chamber;

FIG. 55 is an enlarged view of the humidifier illustrated in Inset AGshown in FIG. 6;

FIG. 56 is an enlarged view of Inset AD shown in FIG. 52;

FIG. 57 is an exploded perspective view of the LOC device within InsetAD;

FIG. 58 is a diagram of a FRET probe in a closed configuration;

FIG. 59 is a diagram of a FRET probe in an open and hybridizedconfiguration;

FIG. 60 is a graph of the intensity of an excitation light over time;

FIG. 61 is a diagram of the excitation illumination geometry of thehybridization chamber array;

FIG. 62 is a diagram of a Sensor Electronic Technology LED illuminationgeometry;

FIG. 63 is an enlarged plan view of the humidity sensor shown in InsetAH of FIG. 6;

FIG. 64 shows the features of a first variant of a thermal bend actuatedvalve;

FIG. 65 is a schematic section view of the first variant of a thermalbend actuated valve taken along line 70-70 of FIG. 64;

FIG. 66 shows the features of a second variant of a thermal bendactuated valve;

FIG. 67 is a schematic section view of the second variant of a thermalbend actuated valve taken along line 72-72 of FIG. 66;

FIG. 68 shows the features of a third variant of a thermal bend actuatedvalve;

FIG. 69 is a schematic section view of the third variant of a thermalbend actuated valve taken along line 74-74 of FIG. 68;

FIG. 70 shows the features of a fault tolerant valve array;

FIG. 71 is a schematic section view of the fault tolerant valve arraytaken along line 76-76 of FIG. 70;

FIG. 72 is a schematic section view of a leukocyte target dialysissection;

FIG. 73 is a schematic showing part of the photodiode array of the photosensor;

FIG. 74 is a circuit diagram for a single photodiode;

FIG. 75 is a timing diagram for the photodiode control signals;

FIG. 76 is an enlarged view of the evaporator shown in Inset AP of FIG.55;

FIG. 77 is a schematic section view through a hybridization chamber witha detection photodiode and trigger photodiode;

FIG. 78 is a diagram of linker-primed PCR;

FIG. 79 is a schematic representation of a test module with a lancet;

FIG. 80 shows a boiling-initiated valve array;

FIG. 81 is a perspective of the boiling-initiated valve sectionedthrough line 97-97 shown in FIG. 80;

FIG. 82 is a perspective of LOC variant VII;

FIG. 83 is an exploded top perspective of the cap for LOC variant VIIwith internal channels and reservoirs shown in dotted line;

FIG. 84 is a bottom perspective of the cap for LOC variant VII showingthe cap channels;

FIG. 85 is a plan view of LOC variant VII with features and structuresfrom all layers superimposed on each other;

FIG. 86 is a plan view of LOC variant VII showing the structures of theCMOS+MST device in isolation;

FIG. 87 is a plan view of LOC variant VII with the structures of the capshown in isolation;

FIG. 88 is an enlarged view of Inset BA shown in FIG. 85;

FIG. 89 is an enlarged view of Inset BB shown in FIG. 85;

FIG. 90 is an enlarged view of Inset BC shown in FIG. 85;

FIG. 91 is an enlarged view of Inset BD shown in FIG. 85;

FIG. 92 is an enlarged view of Inset BE shown in FIG. 88;

FIG. 93 is an enlarged view of Inset BF shown in FIG. 92;

FIG. 94 is an enlarged view of Inset BG shown in FIG. 85;

FIG. 95 is an enlarged view of the hybridization and detection sectionof LOC variant VII;

FIG. 96 is a diagrammatic representation of the architecture of LOCvariant VII;

FIG. 97 is a diagrammatic representation of the architecture of LOCvariant VIII;

FIG. 98 is a schematic illustration of the architecture of LOC variantXIV;

FIG. 99 is a schematic illustration of the architecture of LOC variantXLI;

FIG. 100 is a schematic illustration of the architecture of LOC variantXLII;

FIG. 101 is a schematic illustration of the architecture of LOC variantXLIII;

FIG. 102 is a schematic illustration of the architecture of LOC variantXLIV;

FIG. 103 is a schematic illustration of the architecture of LOC variantXLVII;

FIG. 104 is a diagram of a primer-linked, linear fluorescent probeduring the initial round of amplification;

FIG. 105 is a diagram of a primer-linked, linear fluorescent probeduring a subsequent amplification cycle;

FIGS. 106A to 106F diagrammatically illustrate thermal cycling of aprimer-linked fluorescent stem-and-loop probe;

FIG. 107 is a schematic illustration of the excitation LED relative tothe hybridization chamber array and the photodiodes;

FIG. 108 is a schematic illustration of the excitation LED and opticallens for directing light onto the hybridization chamber array of the LOCdevice;

FIG. 109 is a schematic illustration of the excitation LED, opticallens, and optical prisms for directing light onto the hybridizationchamber array of the LOC device;

FIG. 110 is a schematic illustration of the excitation LED, optical lensand mirror arrangement for directing light onto the hybridizationchamber array of the LOC device;

FIG. 111 is a plan view showing all the features superimposed on eachother, and showing the location of Insets DA to DK;

FIG. 112 is an enlarged view of Inset DG shown in FIG. 111;

FIG. 113 is an enlarged view of Inset DH shown in FIG. 111;

FIG. 114 shows one embodiment of the shunt transistor for thephotodiodes;

FIG. 115 shows one embodiment of the shunt transistor for thephotodiodes;

FIG. 116 shows one embodiment of the shunt transistor for thephotodiodes;

FIG. 117 is a circuit diagram of the differential imager;

FIG. 118 schematically illustrates a negative control fluorescent probein its stem-and-loop configuration;

FIG. 119 schematically illustrates the negative control fluorescentprobe of FIG. 118 in its open configuration;

FIG. 120 schematically illustrates a positive control fluorescent probein its stem-and-loop configuration;

FIG. 121 schematically illustrates the positive control fluorescentprobe of FIG. 120 in its open configuration;

FIG. 122 is a plan view of an electroexplosive valve;

FIG. 123 is a plan view of a thermal-bend-actuated bend-and-break valve;

FIG. 124 is a plan view of a dual thermal-bend-actuated bend-and-breakvalve;

FIG. 125 is a plan view of a stiction valve;

FIG. 126 is a plan view of a stiction valve variant;

FIG. 127 is a plan view of a bubble break valve;

FIG. 128 shows a test module and test module reader configured for usewith ECL detection;

FIG. 129 is a schematic overview of the electronic components in thetest module configured for use with ECL detection;

FIG. 130 shows a test module and alternative test module readers;

FIG. 131 shows a test module and test module reader along with thehosting system housing various databases;

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Overview

This overview identifies the main components of a molecular diagnosticsystem that incorporates embodiments of the present invention.Comprehensive details of the system architecture and operation are setout later in the specification.

Referring to FIGS. 1, 2, 3, 128 and 129, the system has the followingtop level components:

Test modules 10 and 11 are the size of a typical USB memory key and verycheap to produce. Test modules 10 and 11 each contain a microfluidicdevice, typically in the form of a lab-on-a-chip (LOC) device 30preloaded with reagents and typically more than 1000 probes for themolecular diagnostic assay (see FIGS. 1 and 128). Test module 10schematically shown in FIG. 1 uses a fluorescence-based detectiontechnique to identify target molecules, while test module 11 in FIG. 128uses an electrochemiluminescence-based detection technique. The LOCdevice 30 has an integrated photosensor 44 for fluorescence orelectrochemiluminescence detection (described in detail below). Bothtest modules 10 and 11 use a standard Micro-USB plug 14 for power, dataand control, both have a printed circuit board (PCB) 57, and both haveexternal power supply capacitors 32 and an inductor 15. The test modules10 and 11 are both single-use only for mass production and distributionin sterile packaging ready for use.

The outer casing 13 has a macroreceptacle 24 for receiving thebiological sample and a removable sterile sealing tape 22, preferablywith a low tack adhesive, to cover the macroreceptacle prior to use. Amembrane seal 408 with a membrane guard 410 forms part of the outercasing 13 to reduce dehumidification within the test module whileproviding pressure relief from small air pressure fluctuations. Themembrane guard 410 protects the membrane seal 408 from damage.

Test module reader 12 powers the test module 10 or 11 via Micro-USB port16. The test module reader 12 can adopt many different forms and aselection of these are described later. The version of the reader 12shown in FIGS. 1, 3 and 128 is a smart phone embodiment. A block diagramof this reader 12 is shown in FIG. 3. Processor 42 runs applicationsoftware from program storage 43. The processor 42 also interfaces withthe display screen 18 and user interface (UI) touch screen 17 andbuttons 19, a cellular radio 21, wireless network connection 23, and asatellite navigation system 25. The cellular radio 21 and wirelessnetwork connection 23 are used for communications. Satellite navigationsystem 25 is used for updating epidemiological databases with locationdata. The location data can, alternatively, be entered manually via thetouch screen 17 or buttons 19. Data storage 27 holds genetic anddiagnostic information, test results, patient information, assay andprobe data for identifying each probe and its array position. Datastorage 27 and program storage 43 may be shared in a common memoryfacility. Application software installed on the test module reader 12provides analysis of results, along with additional test and diagnosticinformation.

To conduct a diagnostic test, the test module 10 (or test module 11) isinserted into the Micro-USB port 16 on the test module reader 12. Thesterile sealing tape 22 is peeled back and the biological sample (in aliquid form) is loaded into the sample macroreceptacle 24. Pressingstart button 20 initiates testing via the application software. Thesample flows into the LOC device 30 and the on-board assay extracts,incubates, amplifies and hybridizes the sample nucleic acids (thetarget) with presynthesized hybridization-responsive oligonucleotideprobes. In the case of test module 10 (which uses fluorescence-baseddetection), the probes are fluorescently labelled and the LED 26 housedin the casing 13 provides the necessary excitation light to inducefluorescence emission from the hybridized probes (see FIGS. 1 and 2). Intest module 11 (which uses electrochemiluminescence (ECL) detection),the LOC device 30 is loaded with ECL probes (discussed above) and theLED 26 is not necessary for generating the luminescent emission.Instead, electrodes 860 and 870 provide the excitation electricalcurrent (see FIG. 129). The emission (fluorescent or luminescent) isdetected using a photosensor 44 integrated into CMOS circuitry of eachLOC device. The detected signal is amplified and converted to a digitaloutput which is analyzed by the test module reader 12. The reader thendisplays the results.

The data may be saved locally and/or uploaded to a network servercontaining patient records. The test module 10 or 11 is removed from thetest module reader 12 and disposed of appropriately.

FIGS. 1, 3 and 128 show the test module reader 12 configured as a mobilephone/smart phone 28. In other forms, the test module reader is alaptop/notebook 101, a dedicated reader 103, an ebook reader 107, atablet computer 109 or desktop computer 105 for use in hospitals,private practices or laboratories (see FIG. 130). The reader caninterface with a range of additional applications such as patientrecords, billing, online databases and multi-user environments. It canalso be interfaced with a range of local or remote peripherals such asprinters and patient smart cards.

Referring to FIG. 131, the data generated by the test module 10 can beused to update, via the reader 12 and network 125, the epidemiologicaldatabases hosted on the hosting system for epidemiological data 111, thegenetic databases hosted on the hosting system for genetic data 113, theelectronic health records hosted on the hosting system for electronichealth records (EHR) 115, the electronic medical records hosted on thehosting system for electronic medical records (EMR) 121, and thepersonal health records hosted on the hosting system for personal healthrecords (PHR) 123. Conversely, the epidemiological data hosted on thehosting system for epidemiological data 111, the genetic data hosted onthe hosting system for genetic data 113, the electronic health recordshosted on the hosting system for electronic health records (EHR) 115,the electronic medical records hosted on the hosting system forelectronic medical records (EMR) 121, and the personal health recordshosted on the hosting system for personal health records (PHR) 123, canbe used to update, via network 125 and the reader 12, the digital memoryin the LOC 30 of the test module 10.

Referring back to FIGS. 1, 2, 128 and 129 the reader 12 uses batterypower in the mobile phone configuration. The mobile phone readercontains all test and diagnostic information preloaded. Data can also beloaded or updated via a number of wireless or contact interfaces toenable communications with peripheral devices, computers or onlineservers. A Micro-USB port 16 is provided for connection to a computer ormains power supply for battery recharge.

FIG. 79 shows an embodiment of the test module 10 used for tests thatonly require a positive or negative result for a particular target, suchas testing whether a person is infected with, for example, H1N1Influenza A virus. Only a purpose built USB power/indicator-only module47 is adequate. No other reader or application software is necessary. Anindicator 45 on the USB power/indicator-only module 47 signals positiveor negative results. This configuration is well suited to massscreening.

Additional items supplied with the system may include a test tubecontaining reagents for pre-treatment of certain samples, along withspatula and lancet for sample collection. FIG. 79 shows an embodiment ofthe test module incorporating a spring-loaded, retractable lancet 390and lancet release button 392 for convenience. A satellite phone can beused in remote areas.

Test Module Electronics

FIGS. 2 and 129 are block diagrams of the electronic components in thetest modules 10 and 11, respectively. The CMOS circuitry integrated inthe LOC device 30 has a USB device driver 36, a controller 34, aUSB-compatible LED driver 29, clock 33, power conditioner 31, RAM 38 andprogram and data flash memory 40. These provide the control and memoryfor the entire test module 10 or 11 including the photosensor 44, thetemperature sensors 170, the liquid sensors 174, and the various heaters152, 154, 182, 234, together with associated drivers 37 and 39 andregisters 35 and 41. Only the LED 26 (in the case of test module 10),external power supply capacitors 32 and the Micro-USB plug 14 areexternal to the LOC device 30. The LOC devices 30 include bond-pads formaking connections to these external components. The RAM 38 and theprogram and data flash memory 40 have the application software and thediagnostic and test information (Flash/Secure storage, e.g. viaencryption) for over 1000 probes. In the case of test module 11configured for ECL detection, there is no LED 26 (see FIGS. 128 and129). Data is encrypted by the LOC device 30 for secure storage andsecure communication with an external device. The LOC devices 30 areloaded with electrochemiluminescent probes and the hybridizationchambers each have a pair of ECL excitation electrodes 860 and 870.

Many types of test modules 10 are manufactured in a number of testforms, ready for off-the-shelf use. The differences between the testforms lie in the on board assay of reagents and probes.

Some examples of infectious diseases rapidly identified with this systeminclude:

-   -   Influenza—Influenza virus A, B, C, Isavirus, Thogotovirus    -   Pneumonia—respiratory syncytial virus (RSV), adenovirus,        metapneumovirus, Streptococcus pneumoniae, Staphylococcus aureus    -   Tuberculosis—Mycobacterium tuberculosis, bovis, africanum,        canetti, and microti    -   Plasmodium falciparum, Toxoplasma gondii and other protozoan        parasites    -   Typhoid—Salmonella enterica serovar typhi    -   Ebola virus    -   Human immunodeficiency virus (HIV)    -   Dengue Fever—Flavivirus    -   Hepatitis (A through E)    -   Hospital acquired infections—for example Clostridium difficile,        Vancomycin resistant Enterococcus, and Methicillin resistant        Staphylococcus aureus    -   Herpes simplex virus (HSV)    -   Cytomegalovirus (CMV)    -   Epstein-Ban virus (EBV)    -   Encephalitis—Japanese Encephalitis virus, Chandipura virus    -   Whooping cough—Bordetella pertussis    -   Measles—paramyxovirus    -   Meningitis—Streptococcus pneumoniae and Neisseria meningitidis    -   Anthrax—Bacillus anthracis

Some examples of genetic disorders identified with this system include:

-   -   Cystic fibrosis    -   Haemophilia    -   Sickle cell disease    -   Tay-Sachs disease    -   Haemochromatosis    -   Cerebral arteriopathy    -   Crohn's disease    -   Polycistic kidney disease    -   Congential heart disease    -   Rett syndrome

A small selection of cancers identified by the diagnostic systeminclude:

-   -   Ovarian    -   Colon carcinoma    -   Multiple endocrine neoplasia    -   Retinoblastoma    -   Turcot syndrome

The above lists are not exhaustive and the diagnostic system can beconfigured to detect a much greater variety of diseases and conditionsusing nucleic acid and proteomic analysis.

Detailed Architecture of System Components LOC Device

The LOC device 30 is central to the diagnostic system. It rapidlyperforms the four major steps of a nucleic acid based moleculardiagnostic assay, i.e. sample preparation, nucleic acid extraction,nucleic acid amplification, and detection, using a microfluidicplatform. The LOC device also has alternative uses, and these aredetailed later. As discussed above, test modules 10 and 11 can adoptmany different configurations to detect different targets Likewise, theLOC device 30 has numerous different embodiments tailored to thetarget(s) of interest. One form of the LOC device 30 is LOC device 301for fluorescent detection of target nucleic acid sequences in thepathogens of a whole blood sample. For the purposes of illustration, thestructure and operation of LOC device 301 is now described in detailwith reference to FIGS. 4 to 26 and 27 to 57.

FIG. 4 is a schematic representation of the architecture of the LOCdevice 301. For convenience, process stages shown in FIG. 4 areindicated with the reference numeral corresponding to the functionalsections of the LOC device 301 that perform that process stage. Theprocess stages associated with each of the major steps of a nucleic acidbased molecular diagnostic assay are also indicated: sample input andpreparation 288, extraction 290, incubation 291, amplification 292 anddetection 294. The various reservoirs, chambers, valves and othercomponents of the LOC device 301 will be described in more detail later.

FIG. 5 is a perspective view of the LOC device 301. It is fabricatedusing high volume CMOS and MST (microsystems technology) manufacturingtechniques. The laminar structure of the LOC device 301 is illustratedin the schematic (not to scale) partial section view of FIG. 12. The LOCdevice 301 has a silicon substrate 84 which supports the CMOS+MST chip48, comprising CMOS circuitry 86 and an MST layer 87, with a cap 46overlaying the MST layer 87. For the purposes of this patentspecification, the term ‘MST layer’ is a reference to a collection ofstructures and layers that process the sample with various reagents.Accordingly, these structures and components are configured to defineflow-paths with characteristic dimensions that will support capillarydriven flow of liquids with physical characteristics similar to those ofthe sample during processing. In light of this, the MST layer andcomponents are typically fabricated using surface micromachiningtechniques and/or bulk micromachining techniques. However, otherfabrication methods can also produce structures and componentsdimensioned for capillary driven flows and processing very smallvolumes. The specific embodiments described in this specification showthe MST layer as the structures and active components supported on theCMOS circuitry 86, but excluding the features of the cap 46. However,the skilled addressee will appreciate that the MST layer need not haveunderlying CMOS or indeed an overlying cap in order for it to processthe sample.

The overall dimensions of the LOC device shown in the following figuresare 1760 μm×5824 μm. Of course, LOC devices fabricated for differentapplications may have different dimensions.

FIG. 6 shows the features of the MST layer 87 superimposed with thefeatures of the cap. Insets AA to AD, AG and AH shown in FIG. 6 areenlarged in FIGS. 13, 14, 35, 56, 55 and 63, respectively, and describedin detail below for a comprehensive understanding of each structurewithin the LOC device 301. FIGS. 7 to 10 show the features of the cap 46in isolation while FIG. 11 shows the CMOS+MST device 48 structures inisolation.

Laminar Structure

FIGS. 12 and 22 are sketches that diagrammatically show the laminarstructure of the CMOS+MST device 48, the cap 46 and the fluidicinteraction between the two. The figures are not to scale for thepurposes of illustration. FIG. 12 is a schematic section view throughthe sample inlet 68 and FIG. 22 is a schematic section through thereservoir 54. As best shown in FIG. 12, the CMOS+MST device 48 has asilicon substrate 84 which supports the CMOS circuitry 86 that operatesthe active elements within the MST layer 87 above. A passivation layer88 seals and protects the CMOS layer 86 from the fluid flows through theMST layer 87.

Fluid flows through both the cap channels 94 and the MST channels 90(see for example FIGS. 7 and 16) in the cap layer 46 and MST channellayer 100, respectively. Cell transport occurs in the larger channels 94fabricated in the cap 46, while biochemical processes are carried out inthe smaller MST channels 90. Cell transport channels are sized so as tobe able to transport cells in the sample to predetermined sites in theMST channels 90. Transportation of cells with sizes greater than 20microns (for example, certain leukocytes) requires channel dimensionsgreater than 20 microns, and therefore a cross sectional area transverseto the flow of greater than 400 square microns. MST channels,particularly at locations in the LOC where transport of cells is notrequired, can be significantly smaller.

It will be appreciated that cap channel 94 and MST channel 90 aregeneric references and particular MST channels 90 may also be referredto as (for example) heated microchannels or dialysis MST channels inlight of their particular function. MST channels 90 are formed byetching through a MST channel layer 100 deposited on the passivationlayer 88 and patterned with photoresist. The MST channels 90 areenclosed by a roof layer 66 which forms the top (with respect to theorientation shown in the figures) of the CMOS+MST device 48.

Despite sometimes being shown as separate layers, the cap channel layer80 and the reservoir layer 78 are formed from a unitary piece ofmaterial. Of course, the piece of material may also be non-unitary. Thispiece of material is etched from both sides in order to form a capchannel layer 80 in which the cap channels 94 are etched and thereservoir layer 78 in which the reservoirs 54, 56, 58, 60 and 62 areetched. Alternatively, the reservoirs and the cap channels are formed bya micromolding process. Both etching and micromolding techniques areused to produce channels with cross sectional areas transverse to theflow as large as 20,000 square microns, and as small as 8 squaremicrons.

At different locations in the LOC device, there can be a range ofappropriate choices for the cross sectional area of the channeltransverse to the flow. Where large quantities of sample, or sampleswith large constituents, are contained in the channel, a cross-sectionalarea of up to 20,000 square microns (for example, a 200 micron widechannel in a 100 micron thick layer) is suitable. Where small quantitiesof liquid, or mixtures without large cells present, are contained in thechannel, a very small cross sectional area transverse to the flow ispreferable.

A lower seal 64 encloses the cap channels 94 and the upper seal layer 82encloses the reservoirs 54, 56, 58, 60 and 62.

The five reservoirs 54, 56, 58, 60 and 62 are preloaded withassay-specific reagents. In the embodiment described here, thereservoirs are preloaded with the following reagents, but other reagentscan easily be substituted:

-   -   reservoir 54: anticoagulant with option to include erythrocyte        lysis buffer    -   reservoir 56: lysis reagent    -   reservoir 58: restriction enzymes, ligase and linkers (for        linker-primed PCR (see FIG. 78, extracted from T. Stachan et        al., Human Molecular Genetics 2, Garland Science, N.Y. and        London, 1999))    -   reservoir 60: amplification mix (dNTPs, primers, buffer) and    -   reservoir 62: DNA polymerase.

The cap 46 and the CMOS+MST layers 48 are in fluid communication viacorresponding openings in the lower seal 64 and the roof layer 66. Theseopenings are referred to as uptakes 96 and downtakes 92 depending onwhether fluid is flowing from the MST channels 90 to the cap channels 94or vice versa.

LOC Device Operation

The operation of the LOC device 301 is described below in a step-wisefashion with reference to analysing pathogenic DNA in a blood sample. Ofcourse, other types of biological or non-biological fluid are alsoanalysed using an appropriate set, or combination, of reagents, testprotocols, LOC variants and detection systems. Referring back to FIG. 4,there are five major steps involved in analysing a biological sample,comprising sample input and preparation 288, nucleic acid extraction290, nucleic acid incubation 291, nucleic acid amplification 292 anddetection and analysis 294.

The sample input and preparation step 288 involves mixing the blood withan anticoagulant 116 and then separating pathogens from the leukocytesand erythrocytes with the pathogen dialysis section 70. As best shown inFIGS. 7 and 12, the blood sample enters the device via the sample inlet68. Capillary action draws the blood sample along the cap channel 94 tothe reservoir 54. Anticoagulant is released from the reservoir 54 as thesample blood flow opens its surface tension valve 118 (see FIGS. 15 and22). The anticoagulant prevents the formation of clots which would blockthe flow.

As best shown in FIG. 22, the anticoagulant 116 is drawn out of thereservoir 54 by capillary action and into the MST channel 90 via thedowntake 92. The downtake 92 has a capillary initiation feature (CIF)102 to shape the geometry of the meniscus such that it does not anchorto the rim of the downtake 92. Vent holes 122 in the upper seal 82allows air to replace the anticoagulant 116 as it is drawn out of thereservoir 54.

The MST channel 90 shown in FIG. 22 is part of a surface tension valve118. The anticoagulant 116 fills the surface tension valve 118 and pinsa meniscus 120 to the uptake 96 to a meniscus anchor 98. Prior to use,the meniscus 120 remains pinned at the uptake 96 so the anticoagulantdoes not flow into the cap channel 94. When the blood flows through thecap channel 94 to the uptake 96, the meniscus 120 is removed and theanticoagulant is drawn into the flow.

FIGS. 15 to 21 show Inset AE which is a portion of Inset AA shown inFIG. 13. As shown in FIGS. 15, 16 and 17, the surface tension valve 118has three separate MST channels 90 extending between respectivedowntakes 92 and uptakes 96. The number of MST channels 90 in a surfacetension valve can be varied to change the flow rate of the reagent intothe sample mixture. As the sample mixture and the reagents mix togetherby diffusion, the flow rate out of the reservoir determines theconcentration of the reagent in the sample flow. Hence, the surfacetension valve for each of the reservoirs is configured to match thedesired reagent concentration.

The blood passes into a pathogen dialysis section 70 (see FIGS. 4 and15) where target cells are concentrated from the sample using an arrayof apertures 164 sized according to a predetermined threshold. Cellssmaller than the threshold pass through the apertures while larger cellsdo not pass through the apertures. Unwanted cells, which may be eitherthe larger cells withheld by the array of apertures 164 or the smallercells that pass through the apertures, are redirected to a waste unit 76while the target cells continue as part of the assay.

In the pathogen dialysis section 70 described here, the pathogens fromthe whole blood sample are concentrated for microbial DNA analysis. Thearray of apertures is formed by a multitude of 3 micron diameter holes164 fluidically connecting the input flow in the cap channel 94 to atarget channel 74. The 3 micron diameter apertures 164 and the dialysisuptake holes 168 for the target channel 74 are connected by a series ofdialysis MST channels 204 (best shown in FIGS. 15 and 21). Pathogens aresmall enough to pass through the 3 micron diameter apertures 164 andfill the target channel 74 via the dialysis MST channels 204. Cellslarger than 3 microns, such as erythrocytes and leukocytes, stay in thewaste channel 72 in the cap 46 which leads to a waste reservoir 76 (seeFIG. 7).

Other aperture shapes, sizes and aspect ratios can be used to isolatespecific pathogens or other target cells such as leukocytes for humanDNA analysis. Greater detail on the dialysis section and dialysisvariants is provided later.

Referring again to FIGS. 6 and 7, the flow is drawn through the targetchannel 74 to the surface tension valve 128 of the lysis reagentreservoir 56. The surface tension valve 128 has seven MST channels 90extending between the lysis reagent reservoir 56 and the target channel74. When the menisci are unpinned by the sample flow, the flow rate fromall seven of the MST channels 90 will be greater than the flow rate fromthe anticoagulant reservoir 54 where the surface tension valve 118 hasthree MST channels 90 (assuming the physical characteristics of thefluids are roughly equivalent). Hence the proportion of lysis reagent inthe sample mixture is greater than that of the anticoagulant.

The lysis reagent and target cells mix by diffusion in the targetchannel 74 within the chemical lysis section 130. A boiling-initiatedvalve 126 stops the flow until sufficient time has passed for diffusionand lysis to take place, releasing the genetic material from the targetcells (see FIGS. 6 and 7). The structure and operation of theboiling-initiated valves are described in greater detail below withreference to FIGS. 31 and 32. Other active valve types (as opposed topassive valves such as the surface tension valve 118) have also beendeveloped by the Applicant which may be used here instead of theboiling-initiated valve. These alternative valve designs are alsodescribed later.

When the boiling-initiated valve 126 opens, the lysed cells flow into amixing section 131 for pre-amplification restriction digestion andlinker ligation.

Referring to FIG. 13, restriction enzymes, linkers and ligase arereleased from the reservoir 58 when the flow unpins the menisci at thesurface tension valve 132 at the start of the mixing section 131. Themixture flows the length of the mixing section 131 for diffusion mixing.At the end of the mixing section 131 is downtake 134 leading into theincubator inlet channel 133 of the incubation section 114 (see FIG. 13).The incubator inlet channel 133 feeds the mixture into a serpentineconfiguration of heated microchannels 210 which provides an incubationchamber for holding the sample during restriction digestion and ligationof the linkers (see FIGS. 13 and 14).

FIGS. 23, 24, 25, 26, 27, 28 and 29 show the layers of the LOC device301 within Inset AB of FIG. 6. Each figure shows the sequential additionof layers forming the structures of the CMOS+MST layer 48 and the cap46. Inset AB shows the end of the incubation section 114 and the startof the amplification section 112. As best shown in FIGS. 14 and 23, theflow fills the microchannels 210 of the incubation section 114 untilreaching the boiling-initiated valve 106 where the flow stops whilediffusion takes place. As discussed above, the microchannel 210 upstreamof the boiling-initiated valve 106 becomes an incubation chambercontaining the sample, restriction enzymes, ligase and linkers. Theheaters 154 are then activated and held at constant temperature for aspecified time for restriction digestion and linker ligation to occur.

The skilled worker will appreciate that this incubation step 291 (seeFIG. 4) is optional and only required for some nucleic acidamplification assay types. Furthermore, in some instances, it may benecessary to have a heating step at the end of the incubation period tospike the temperature above the incubation temperature. The temperaturespike inactivates the restriction enzymes and ligase prior to enteringthe amplification section 112. Inactivation of the restriction enzymesand ligase has particular relevance when isothermal nucleic acidamplification is being employed.

Following incubation, the boiling-initiated valve 106 is activated(opened) and the flow resumes into the amplification section 112.Referring to FIGS. 31 and 32, the mixture fills the serpentineconfiguration of heated microchannels 158, which form one or moreamplification chambers, until it reaches the boiling-initiated valve108. As best shown in the schematic section view of FIG. 30,amplification mix (dNTPs, primers, buffer) is released from reservoir 60and polymerase is subsequently released from reservoir 62 into theintermediate MST channel 212 connecting the incubation and amplificationsections (114 and 112 respectively).

FIGS. 35 to 51 show the layers of the LOC device 301 within Inset AC ofFIG. 6. Each figure shows the sequential addition of layers forming thestructures of the CMOS+MST device 48 and the cap 46. Inset AC is at theend of the amplification section 112 and the start of the hybridizationand detection section 52. The incubated sample, amplification mix andpolymerase flow through the microchannels 158 to the boiling-initiatedvalve 108. After sufficient time for diffusion mixing, the heaters 154in the microchannels 158 are activated for thermal cycling or isothermalamplification. The amplification mix goes through a predetermined numberof thermal cycles or a preset amplification time to amplify sufficienttarget DNA. After the nucleic acid amplification process, theboiling-initiated valve 108 opens and flow resumes into thehybridization and detection section 52. The operation ofboiling-initiated valves is described in more detail later.

As shown in FIG. 52, the hybridization and detection section 52 has anarray of hybridization chambers 110. FIGS. 52, 53, 54 and 56 show thehybridization chamber array 110 and individual hybridization chambers180 in detail. At the entrance to the hybridization chamber 180 is adiffusion barrier 175 which prevents diffusion of the target nucleicacid, probe strands and hybridized probes between the hybridizationchambers 180 during hybridization so as to prevent erroneoushybridization detection results. The diffusion barriers 175 present aflow-path-length that is long enough to prevent the target sequences andprobes diffusing out of one chamber and contaminating another chamberwithin the time taken for the probes and nucleic acids to hybridize andthe signal to be detected, thus avoiding an erroneous result.

Another mechanism to prevent erroneous readings is to have identicalprobes in a number of the hybridization chambers. The CMOS circuitry 86derives a single result from the photodiodes 184 corresponding to thehybridization chambers 180 that contain identical probes. Anomalousresults can be disregarded or weighted differently in the derivation ofthe single result.

The thermal energy required for hybridization is provided byCMOS-controlled heaters 182 (described in more detail below). After theheater is activated, hybridization occurs between complementarytarget-probe sequences. The LED driver 29 in the CMOS circuitry 86signals the LED 26 located in the test module 10 to illuminate. Theseprobes only fluoresce when hybridization has occurred thereby avoidingwashing and drying steps that are typically required to remove unboundstrands. Hybridization forces the stem-and-loop structure of the FRETprobes 186 to open, which allows the fluorophore to emit fluorescentenergy in response to the LED excitation light, as discussed in greaterdetail later. Fluorescence is detected by a photodiode 184 in the CMOScircuitry 86 underlying each hybridization chamber 180 (seehybridization chamber description below). The photodiodes 184 for allhybridization chambers and associated electronics collectively form thephotosensor 44 (see FIG. 73). In other embodiments, the photosensor maybe an array of charge coupled devices (CCD array). The detected signalfrom the photodiodes 184 is amplified and converted to a digital outputwhich is analyzed by the test module reader 12. Further details of thedetection method are described later.

Additional Details for the LOC Device Modularity of the Design

The LOC device 301 has many functional sections, including the reagentreservoirs 54, 56, 58, 60 and 62, the dialysis section 70, lysis section130, incubation section 114, and amplification section 112, valve types,the humidifier and humidity sensor. In other embodiments of the LOCdevice, these functional sections can be omitted, additional functionalsections can be added or the functional sections can be used foralternative purposes to those described above.

For example, the incubation section 114 can be used as the firstamplification section 112 of a tandem amplification assay system, withthe chemical lysis reagent reservoir 56 being used to add the firstamplification mix of primers, dNTPs and buffer and reagent reservoir 58being used for adding the reverse transcriptase and/or polymerase. Achemical lysis reagent can also be added to the reservoir 56 along withthe amplification mix if chemical lysis of the sample is desired or,alternatively, thermal lysis can occur in the incubation section byheating the sample for a predetermined time. In some embodiments, anadditional reservoir can be incorporated immediately upstream ofreservoir 58 for the mix of primers, dNTPs and buffer if there is arequirement for chemical lysis and a separation of this mix from thechemical lysis reagent is desired.

In some circumstances it may be desirable to omit a step, such as theincubation step 291. In this case, a LOC device can be specificallyfabricated to omit the reagent reservoir 58 and incubation section 114,or the reservoir can simply not be loaded with reagents or the activevalves, if present, not activated to dispense the reagents into thesample flow, and the incubation section then simply becomes a channel totransport the sample from the lysis section 130 to the amplificationsection 112. The heaters are independently operable and therefore, wherereactions are dependent on heat, such as thermal lysis, programming theheaters not to activate during this step ensures thermal lysis does notoccur in LOC devices that do not require it. The dialysis section 70 canbe located at the beginning of the fluidic system within themicrofluidic device as shown in FIG. 4 or can be located anywhere elsewithin the microfluidic device. For example, dialysis after theamplification phase 292 to remove cellular debris prior to thehybridization and detection step 294 may be beneficial in somecircumstances. Alternatively, two or more dialysis sections can beincorporated at any location throughout the LOC device. Similarly, it ispossible to incorporate additional amplification sections 112 to enablemultiple targets to be amplified in parallel or in series prior to beingdetected in the hybridization chamber arrays 110 with specific nucleicacid probes. For analysis of samples like whole blood, in which dialysisis not required, the dialysis section 70 is simply omitted from thesample input and preparation section 288 of the LOC design. In somecases, it is not necessary to omit the dialysis section 70 from the LOCdevice even if the analysis does not require dialysis. If there is nogeometric hindrance to the assay by the existence of a dialysis section,a LOC with the dialysis section 70 in the sample input and preparationsection can still be used without a loss of the required functionality.

Furthermore, the detection section 294 may encompass proteomic chamberarrays which are identical to the hybridization chamber arrays but areloaded with probes designed to conjugate or hybridize with sample targetproteins present in non-amplified sample instead of nucleic acid probesdesigned to hybridize to target nucleic acid sequences.

It will be appreciated that the LOC devices fabricated for use in thisdiagnostic system are different combinations of functional sectionsselected in accordance with the particular LOC application. The vastmajority of functional sections are common to many of the LOC devicesand the design of additional LOC devices for new application is a matterof compiling an appropriate combination of functional sections from theextensive selection of functional sections used in the existing LOCdevices.

Only a small number of the LOC devices are shown in this description andsome more are shown schematically to illustrate the design flexibilityof the LOC devices fabricated for this system. The person skilled in theart will readily recognise that the LOC devices shown in thisdescription are not an exhaustive list and many additional LOC designsare a matter of compiling the appropriate combination of functionalsections.

Sample Types

LOC variants can accept and analyze the nucleic acid or protein contentof a variety of sample types in liquid form including, but not limitedto, blood and blood products, saliva, cerebrospinal fluid, urine, semen,amniotic fluid, umbilical cord blood, breast milk, sweat, pleuraleffusion, tear, pericardial fluid, peritoneal fluid, environmental watersamples and drink samples. Amplicon obtained from macroscopic nucleicacid amplification can also be analysed using the LOC device; in thiscase, all the reagent reservoirs will be empty or configured not torelease their contents, and the dialysis, lysis, incubation andamplification sections will be used solely to transport the sample fromthe sample inlet 68 to the hybridization chambers 180 for nucleic aciddetection, as described above.

For some sample types, a pre-processing step is required, for examplesemen may need to be liquefied and mucus may need to be pre-treated withan enzyme to reduce the viscosity prior to input into the LOC device.

Sample Input

Referring to FIGS. 1 and 12, the sample is added to the macroreceptacle24 of the test module 10. The macroreceptacle 24 is a truncated conewhich feeds into the inlet 68 of the LOC device 301 by capillary action.Here it flows into the 64 μm wide×60 μm deep cap channel 94 where it isdrawn towards the anticoagulant reservoir 54, also by capillary action.

Reagent Reservoirs

The small volumes of reagents required by the assay systems usingmicrofluidic devices, such as LOC device 301, permit the reagentreservoirs to contain all reagents necessary for the biochemicalprocessing with each of the reagent reservoirs having a small volume.This volume is easily less than 1,000,000,000 cubic microns, in the vastmajority of cases less than 300,000,000 cubic microns, typically lessthan 70,000,000 cubic microns and in the case of the LOC device 301shown in the drawings, less than 20,000,000 cubic microns.

Dialysis Section

Referring to FIGS. 15 to 21, 33 and 34, the pathogen dialysis section 70is designed to concentrate pathogenic target cells from the sample. Aspreviously described, a plurality of apertures in the form of 3 microndiameter holes 164 in the roof layer 66 filter the target cells from thebulk of the sample. As the sample flows past the 3 micron diameterapertures 164, microbial pathogens pass through the holes into a seriesof dialysis MST channels 204 and flow back up into the target channel 74via 16 μm dialysis uptake holes 168 (see FIGS. 33 and 34). The remainderof the sample (erythrocytes and so on) stay in the cap channel 94.Downstream of the pathogen dialysis section 70, the cap channel 94becomes the waste channel 72 leading to the waste reservoir 76. Forbiological samples of the type that generate a substantial amount ofwaste, a foam insert or other porous element 49 within the outer casing13 of the test module 10 is configured to be in fluid communication withthe waste reservoir 76 (see FIG. 1).

The pathogen dialysis section 70 functions entirely on capillary actionof the fluid sample. The 3 micron diameter apertures 164 at the upstreamend of the pathogen dialysis section 70 have capillary initiationfeatures (CIFs) 166 (see FIG. 33) so that the fluid is drawn down intothe dialysis MST channel 204 beneath. The first uptake hole 198 for thetarget channel 74 also has a CIF 202 (see FIG. 15) to avoid the flowsimply pinning a meniscus across the dialysis uptake holes 168.

The small constituents dialysis section 682 schematically shown in FIG.99 can have a similar structure to the pathogen dialysis section 70. Thesmall constituents dialysis section separates any small target cells ormolecules from a sample by sizing (and, if necessary, shaping) aperturessuitable for allowing the small target cells or molecules to pass intothe target channel and continue for further analysis. Larger sized cellsor molecules are removed to a waste reservoir 766. Thus, the LOC device30 (see FIGS. 1 and 128) is not limited to separating pathogens that areless than 3 μm in size, but can be used to separate cells or moleculesof any size desired.

Lysis Section

Referring back to FIGS. 7, 11 and 13, the genetic material in the sampleis released from the cells by a chemical lysis process. As describedabove, a lysis reagent from the lysis reservoir 56 mixes with the sampleflow in the target channel 74 downstream of the surface tension valve128 for the lysis reservoir 56. However, some diagnostic assays arebetter suited to a thermal lysis process, or even a combination ofchemical and thermal lysis of the target cells. The LOC device 301accommodates this with the heated microchannels 210 of the incubationsection 114. The sample flow fills the incubation section 114 and stopsat the boiling-initiated valve 106. The incubation microchannels 210heat the sample to a temperature at which the cellular membranes aredisrupted.

In some thermal lysis applications, an enzymatic reaction in thechemical lysis section 130 is not necessary and the thermal lysiscompletely replaces the enzymatic reaction in the chemical lysis section130.

Boiling-Initiated Valve As discussed above, the LOC device 301 has threeboiling-initiated valves 126, 106 and 108. The location of these valvesis shown in FIG. 6. FIG. 31 is an enlarged plan view of theboiling-initiated valve 108 in isolation at the end of the heatedmicrochannels 158 of the amplification section 112.

The sample flow 119 is drawn along the heated microchannels 158 bycapillary action until it reaches the boiling-initiated valve 108. Theleading meniscus 120 of the sample flow pins at a meniscus anchor 98 atthe valve inlet 146. The geometry of the meniscus anchor 98 stops theadvancing meniscus to arrest the capillary flow. As shown in FIGS. 31and 32, the meniscus anchor 98 is an aperture provided by an uptakeopening from the MST channel 90 to the cap channel 94. Surface tensionin the meniscus 120 keeps the valve closed. An annular heater 152 is atthe periphery of the valve inlet 146. The annular heater 152 isCMOS-controlled via the boiling-initiated valve heater contacts 153.

To open the valve, the CMOS circuitry 86 sends an electrical pulse tothe valve heater contacts 153. The annular heater 152 resistively heatsuntil the liquid sample 119 boils. The boiling unpins the meniscus 120from the valve inlet 146 and initiates wetting of the cap channel 94.Once wetting the cap channel 94 begins, capillary flow resumes. Thefluid sample 119 fills the cap channel 94 and flows through the valvedowntake 150 to the valve outlet 148 where capillary driven flowcontinues along the amplification section exit channel 160 into thehybridization and detection section 52. Liquid sensors 174 are placedbefore and after the valve for diagnostics.

It will be appreciated that once the boiling-initiated valves areopened, they cannot be re-closed. However, as the LOC device 301 and thetest module 10 are single-use devices, re-closing the valves isunnecessary.

Incubation Section and Nucleic Acid Amplification Section

FIGS. 6, 7, 13, 14, 23, 24, 25, 35 to 45, 50 and 51 show the incubationsection 114 and the amplification section 112. The incubation section114 has a single, heated incubation microchannel 210 etched in aserpentine pattern in the MST channel layer 100 from the downtakeopening 134 to the boiling-initiated valve 106 (see FIGS. 13 and 14).Control over the temperature of the incubation section 114 enablesenzymatic reactions to take place with greater efficiency. Similarly,the amplification section 112 has a heated amplification microchannel158 in a serpentine configuration leading from the boiling-initiatedvalve 106 to the boiling-initiated valve 108 (see FIGS. 6 and 14). Thesevalves arrest the flow to retain the target cells in the heatedincubation or amplification microchannels 210 or 158 while mixing,incubation and nucleic acid amplification takes place. The serpentinepattern of the microchannels also facilitates (to some extent) mixing ofthe target cells with reagents.

In the incubation section 114 and the amplification section 112, thesample cells and the reagents are heated by the heaters 154 controlledby the CMOS circuitry 86 using pulse width modulation (PWM). Eachmeander of the serpentine configuration of the heated incubationmicrochannel 210 and amplification microchannel 158 has three separatelyoperable heaters 154 extending between their respective heater contacts156 (see FIG. 14) which provides for the two-dimensional control ofinput heat flux density. As best shown in FIG. 51, the heaters 154 aresupported on the roof layer 66 and embedded in the lower seal 64. Theheater material is TiAl but many other conductive metals would besuitable. The elongate heaters 154 are parallel with the longitudinalextent of each channel section that forms the wide meanders of theserpentine shape. In the amplification section 112, each of the widemeanders can operate as separate PCR chambers via individual heatercontrol.

The small volumes of amplicon required by the assay systems usingmicrofluidic devices, such as LOC device 301, permit low amplificationmixture volumes for amplification in amplification section 112. Thisvolume is easily less than 400 nanoliters, in the vast majority of casesless than 170 nanoliters, typically less than 70 nanoliters and in thecase of the LOC device 301, between 2 nanoliters and 30 nanoliters.

Increased Rates of Heating and Greater Diffusive Mixing

The small cross section of each channel section increases the heatingrate of the amplification fluid mix. All the fluid is kept a relativelyshort distance from the heater 154. Reducing the channel cross section(that is the amplification microchannel 158 cross section) to less than100,000 square microns achieves appreciably higher heating rates thanthat provided by more ‘macro-scale’ equipment. Lithographic fabricationtechniques allow the amplification microchannel 158 to have a crosssectional area transverse to the flow-path less than 16,000 squaremicrons which gives substantially higher heating rates. Feature sizes onthe order of 1 micron are readily achievable with lithographictechniques. If very little amplicon is needed (as is the case in the LOCdevice 301), the cross sectional area can be reduced to less than 2,500square microns. For diagnostic assays with 1,000 to 2,000 probes on theLOC device, and a requirement of ‘sample-in, answer out’ in less than 1minute, a cross sectional area transverse to the flow of between 400square microns and 1 square micron is adequate.

The heater element in the amplification microchannel 158 heats thenucleic acid sequences at a rate more than 80 Kelvin (K) per second, inthe vast majority of cases at a rate greater than 100 K per second.Typically, the heater element heats the nucleic acid sequences at a ratemore than 1,000 K per second and in many cases, the heater element heatsthe nucleic acid sequences at a rate more than 10,000 K per second.Commonly, based on the demands of the assay system, the heater elementheats the nucleic acid sequences at a rate more than 100,000 K persecond, more than 1,000,000 K per second more than 10,000,000 K persecond, more than 20,000,000 K per second, more than 40,000,000 K persecond, more than 80,000,000 K per second and more than 160,000,000 Kper second.

A small cross-sectional area channel is also beneficial for diffusivemixing of any reagents with the sample fluid. Before diffusive mixing iscomplete, diffusion of one liquid into the other is greatest near theinterface between the two. Concentration decreases with distance fromthe interface. Using microchannels with relatively small cross sectionstransverse to the flow direction, keeps both fluid flows close to theinterface for more rapid diffusive mixing. Reducing the channel crosssection to less than 100,000 square microns achieves appreciably highermixing rates than that provided by more ‘macro-scale’ equipment.Lithographic fabrication techniques allows microchannels with a crosssectional area transverse to the flow-path less than 16000 squaremicrons which gives significantly higher mixing rates. If small volumesare needed (as is the case in the LOC device 301), the cross sectionalarea can be reduced to less than 2500 square microns. For diagnosticassays with 1000 to 2000 probes on the LOC device, and a requirement of‘sample-in, answer out’ in less than 1 minute, a cross sectional areatransverse to the flow of between 400 square microns and 1 square micronis adequate.

Short Thermal Cycle Times

Keeping the sample mixture proximate to the heaters, and using verysmall fluid volumes allows rapid thermal cycling during the nucleic acidamplification process. Each thermal cycle (i.e. denaturing, annealingand primer extension) is completed in less than 30 seconds for targetsequences up to 150 base pairs (bp) long. In the vast majority ofdiagnostic assays, the individual thermal cycle times are less than 11seconds, and a large proportion are less than 4 seconds. LOC devices 30with some of the most common diagnostic assays have thermal cycles timebetween 0.45 seconds to 1.5 seconds for target sequences up to 150 bplong. Thermal cycling at this rate allows the test module to completethe nucleic acid amplification process in much less than 10 minutes;often less than 220 seconds. For most assays, the amplification sectiongenerates sufficient amplicon in less than 80 seconds from the samplefluid entering the sample inlet. For a great many assays, sufficientamplicon is generated in 30 seconds.

Upon completion of a preset number of amplification cycles, the ampliconis fed into the hybridization and detection section 52 via theboiling-initiated valve 108.

Hybridization Chambers

FIGS. 52, 53, 54, 56 and 57 show the hybridization chambers 180 in thehybridization chamber array 110. The hybridization and detection section52 has a 24×45 array 110 of hybridization chambers 180, each withhybridization-responsive FRET probes 186, heater element 182 and anintegrated photodiode 184. The photodiode 184 is incorporated fordetection of fluorescence resulting from the hybridization of a targetnucleic acid sequence or protein with the FRET probes 186. Eachphotodiode 184 is independently controlled by the CMOS circuitry 86. Anymaterial between the FRET probes 186 and the photodiode 184 must betransparent to the emitted light. Accordingly, the wall section 97between the probes 186 and the photodiode 184 is also opticallytransparent to the emitted light. In the LOC device 301, the wallsection 97 is a thin (approximately 0.5 micron) layer of silicondioxide.

Incorporation of a photodiode 184 directly beneath each hybridizationchamber 180 allows the volume of probe-target hybrids to be very smallwhile still generating a detectable fluorescence signal (see FIG. 54).The small amounts permit small volume hybridization chambers. Adetectable amount of probe-target hybrid requires a quantity of probe,prior to hybridization, which is easily less than 270 picograms(corresponding to 900,000 cubic microns), in the vast majority of casesless than 60 picograms (corresponding to 200,000 cubic microns),typically less than 12 picograms (corresponding to 40,000 cubic microns)and in the case of the LOC device 301 shown in the accompanying figures,less than 2.7 picograms (corresponding to a chamber volume of 9,000cubic microns). Of course, reducing the size of the hybridizationchambers allows a higher density of chambers and therefore more probeson the LOC device. In LOC device 301, the hybridization section has morethan 1,000 chambers in an area of 1,500 microns by 1,500 microns (i.e.less than 2,250 square microns per chamber). Smaller volumes also reducethe reaction times so that hybridization and detection is faster. Anadditional advantage of the small amount of probe required in eachchamber is that only very small quantities of probe solution need to bespotted into each chamber during production of the LOC device.Embodiments of the LOC device according to the invention can be spottedusing a probe solution volume of 1 picoliter or less.

After nucleic acid amplification, boiling-initiated valve 108 isactivated and the amplicon flows along the flow-path 176 and into eachof the hybridization chambers 180 (see FIGS. 52 and 56). An end-pointliquid sensor 178 indicates when the hybridization chambers 180 arefilled with amplicon and the heaters 182 can be activated.

After sufficient hybridization time, the LED 26 (see FIG. 2) isactivated. The opening in each of the hybridization chambers 180provides an optical window 136 for exposing the FRET probes 186 to theexcitation radiation (see FIGS. 52, 54 and 56). The LED 26 isilluminated for a sufficiently long time in order to induce afluorescence signal from the probes with high intensity. Duringexcitation, the photodiode 184 is shorted. After a pre-programmed delay300 (see FIG. 2), the photodiode 184 is enabled and fluorescenceemission is detected in the absence of the excitation light. Theincident light on the active area 185 of the photodiode 184 (see FIG.54) is converted into a photocurrent which can then be measured usingCMOS circuitry 86.

The hybridization chambers 180 are each loaded with probes for detectinga single target nucleic acid sequence. Each hybridization chambers 180can be loaded with probes to detect over 1,000 different targets ifdesired. Alternatively, many or all the hybridization chambers can beloaded with the same probes to detect the same target nucleic acidrepeatedly. Replicating the probes in this way throughout thehybridization chamber array 110 leads to increased confidence in theresults obtained and the results can be combined by the photodiodesadjacent those hybridization chambers to provide a single result ifdesired. The person skilled in the art will recognise that it ispossible to have from one to over 1,000 different probes on thehybridization chamber array 110, depending on the assay specification.

Humidifier and Humidity Sensor

Inset AG of FIG. 6 indicates the position of the humidifier 196. Thehumidifier prevents evaporation of the reagents and probes duringoperation of the LOC device 301. As best shown in the enlarged view ofFIG. 55, a water reservoir 188 is fluidically connected to threeevaporators 190. The water reservoir 188 is filled with molecularbiology-grade water and sealed during manufacturing. As best shown inFIGS. 55 and 76, water is drawn into three downtakes 194 and alongrespective water supply channels 192 by capillary action to a set ofthree uptakes 193 at the evaporators 190. A meniscus pins at each uptake193 to retain the water. The evaporators have annular shaped heaters 191which encircle the uptakes 193. The annular heaters 191 are connected tothe CMOS circuitry 86 by the conductive columns 376 to the top metallayer 195 (see FIG. 37). Upon activation, the annular heaters 191 heatthe water causing evaporation and humidifying the device surrounds.

The position of the humidity sensor 232 is also shown in FIG. 6.However, as best shown in the enlarged view of Inset AH in FIG. 63, thehumidity sensor has a capacitive comb structure. A lithographicallyetched first electrode 296 and a lithographically etched secondelectrode 298 face each other such that their teeth are interleaved. Theopposed electrodes form a capacitor with a capacitance that can bemonitored by the CMOS circuitry 86. As the humidity increases, thepermittivity of the air gap between the electrodes increases, so thatthe capacitance also increases. The humidity sensor 232 is adjacent thehybridization chamber array 110 where humidity measurement is mostimportant to slow evaporation from the solution containing the exposedprobes.

Feedback Sensors

Temperature and liquid sensors are incorporated throughout the LOCdevice 301 to provide feedback and diagnostics during device operation.Referring to FIG. 35, nine temperature sensors 170 are distributedthroughout the amplification section 112. Likewise, the incubationsection 114 also has nine temperature sensors 170. These sensors eachuse a 2×2 array of bipolar junction transistors (BJTs) to monitor thefluid temperature and provide feedback to the CMOS circuitry 86. TheCMOS circuitry 86 uses this to precisely control the thermal cyclingduring the nucleic acid amplification process and any heating duringthermal lysis and incubation.

In the hybridization chambers 180, the CMOS circuitry 86 uses thehybridization heaters 182 as temperature sensors (see FIG. 56). Theelectrical resistance of the hybridization heaters 182 is temperaturedependent and the CMOS circuitry 86 uses this to derive a temperaturereading for each of the hybridization chambers 180.

The LOC device 301 also has a number of MST channel liquid sensors 174and cap channel liquid sensors 208. FIG. 35 shows a line of MST channelliquid sensors 174 at one end of every other meander in the heatedmicrochannel 158. As best shown in FIG. 37, the MST channel liquidsensors 174 are a pair of electrodes formed by exposed areas of the topmetal layer 195 in the CMOS structure 86. Liquid closes the circuitbetween the electrodes to indicate its presence at the sensor'slocation.

FIG. 25 shows an enlarged perspective of cap channel liquid sensors 208.Opposing pairs of TiAl electrodes 218 and 220 are deposited on the rooflayer 66. Between the electrodes 218 and 220 is a gap 222 to hold thecircuit open in the absence of liquid. The presence of liquid closes thecircuit and the CMOS circuitry 86 uses this feedback to monitor theflow.

Gravitational Independence

The test modules 10 are orientation independent. They do not need to besecured to a flat stable surface in order to operate. Capillary drivenfluid flows and a lack of external plumbing into ancillary equipmentallow the modules to be truly portable and simply plugged into asimilarly portable hand held reader such as a mobile telephone. Having agravitationally independent operation means the test modules are alsoaccelerationally independent to all practical extents. They areresistant to shock and vibration and will operate on moving vehicles orwhile the mobile telephone is being carried around.

Valve Alternatives Thermal Bend Actuated Valve Variant 1

FIGS. 64 and 65 show a first variant 302 of a thermal bend actuatedvalve which is a thermal bend actuated pressure pulse valve. FIG. 65 isa schematic section view through line 70-70 shown in FIG. 64. The firstvariant thermal bend actuated valve 302 has movable member in the formof a CMOS-activated thermal bend actuator 304 of TiAl, TiN or similarresistive heater material. The sample flow enters the valve inlets 146in the MST channel 90 but stops when a meniscus 120 pins to the aperture306. The embodiment shown in FIGS. 64 and 65 shows the aperture externalto the movable member, but it may also be defined at least partially bythe movable member. In this state, the valve is closed. To open thevalve, the CMOS circuitry 86 sends a series of electrical pulses to thethermal bend actuator 304. The CMOS-activated thermal bend actuator 304is bonded to a cantilevered section 162 of the roof layer 66 fixed atthe inlet end and free at the aperture end. Differential thermalexpansion bends the cantilevered section 162 such that it moves rapidlytowards the passivation layer 88. Fluidic drag prevents backflow of theliquid sample 119 which instead, is ejected through the aperture 306into the cap channel 94. Reciprocating the cantilevered section 162between the quiescent and displaced positions for a period of timeensures the meniscus 120 unpins from the aperture 306. Sample 119accumulates in the cap channel 94 until its surfaces are wetted andcapillary driven flow resumes. The sample fills the cap channel 94 andthen flows to the valve outlet 148 through the valve downtake 150.

Thermal Bend Actuated Valve Variant 2

FIGS. 66 and 67 show a second variant of the thermal bend actuated valve308 which is a thermal bend actuated surface tension valve. FIG. 67 is aschematic section view through line 72-72 shown in FIG. 66. The secondvariant of the thermal bend actuated valve 308 has a CMOS-activatedthermal bend actuator 304 of TiAl, TiN or similar resistive heatermaterial. The sample flows along the MST channel 90 and into the valveinlet 146 provided by the valve uptake 151. The liquid sample 119 fillsthe cap channel 94 but stops when a meniscus 120 pins to the aperture306. In this state, the valve is closed. To open the valve, the CMOScircuitry 86 sends a series of electrical pulses to the thermal bendactuator 304. The CMOS-activated thermal bend actuator 304 is bonded toa cantilevered section 162 of the roof layer 66 fixed at the inlet endand free at the aperture end. Differential thermal expansion bends thecantilevered section 162 such that the aperture 306 moves towards thepassivation layer 88. The aperture 306 drags the meniscus 120 from thecap channel 94 into the MST channel 90 to re-establish capillary flow.To re-establish the capillary flow, the channel has opposing side wallsthat converge to a narrow section immediately downstream of the movablemember such that the meniscus contacts the narrow section when themovable member moves to the actuated position. Liquid sensors 174 areplaced before and after the valve for diagnostics.

Thermal Bend Actuated Valve Variant 3

FIGS. 68 and 69 show a third variant of the thermal bend actuated valve312, which is a thermal bend actuated surface tension valve, and is usedwhen the liquid sample is to be retained in the cap channel 94. FIG. 69is a schematic section view through line 74-74 shown in FIG. 68. Thethird variant of the thermal bend actuated valve 312 is similar to thesecond variant of the thermal bend actuated valve 308 with the exceptionthat the valve inlet 146 is in a cap channel 94. The sample flows alongthe cap channel 94 and into the valve inlet 146 immediately upstream ofthe CMOS-activated thermal bend actuator 304. The liquid sample 119fills the cap channel 94 but stops when a meniscus 120 pins to theaperture 306. In this state, the valve is closed. To open the valve, theCMOS circuitry 86 sends a series of electrical pulses to the thermalbend actuator 304. The thermal bend actuator 304 is bonded to acantilevered section 162 of the roof layer 66 fixed at the inlet end andfree at the aperture end. Differential thermal expansion bends thecantilevered section 162 such that the aperture 306 moves towards thepassivation layer 88. The aperture 306 drags the meniscus 120 from thecap channel 94 into the MST channel 90 to re-establish capillary flowalong the valve outlet 148. A liquid sensor 174 is placed after thevalve for diagnostics.

Fault-Tolerant Multiple Valve Array

FIG. 70 shows a fault tolerant multiple valve array 314 that may be usedinstead of any of the valve variants 108, 302, 308 and 312 describedabove. FIG. 71 is a schematic section view of the fault tolerantmultiple valve array 314 taken through line 76-76 of FIG. 70.

In the valve variants described above, there is a risk that the sampleflow will fail to pin at the aperture 306 (or valve inlet 146 in thecase of the boiling-initiated valves) and the capillary flow simplycontinues to the valve outlet 148. In effect, the valve fails to close.Conversely, the valve may fail to open (that is, the meniscus does notunpin when the valve actuates). To address this, the fault tolerantmultiple valve array 314 provides fault-tolerance that will allow one ormore individual valve failures.

The fault tolerant multiple valve array 314 has four individual valves;first valve 316, second valve 318, third valve 320 and fourth valve 322.All valves are thermal bend actuated valves of the second variant 308type (thermal bend actuated surface tension valve) described above. Afirst flow-path 324 and a second flow-path 326 extend between the valveinlet 146 and valve outlet 148. The first and second valves (316 and318) are positioned along the first flow-path 324 and the third andfourth valves (320 and 322) are on the second flow-path 326.

Liquid sensors 174 are positioned at the valve inlet 146 and the valveoutlet 148, and between the two valves in each flow-path. The sensorsregister which, if any, of the first, second, third or fourth valvesfail. If the first or the third valves (316 and 320) fail to pin ameniscus, the sample 119 flow is stopped by the second and fourth valves(318 and 322). Similarly, if the meniscus fails to unpin duringactuation of the first and third valves, an alternative flow-path isavailable. The fault tolerant multiple valve array 314 only fails in theunlikely event that both valves in the first or second flow-paths (324or 326) fail. For greater fault tolerance, the fault tolerant multiplevalve array 314 is arbitrarily expandable in terms of the number offluid flow-paths and the number of valves on each flow-path.

Boiling-Initiated Valve Array

FIGS. 80 and 81 show another variant of the fault tolerant multiplevalve array 448 in which the four individual valves are allboiling-initiated valves. As with the mechanical fault tolerant valvearray 314, there is a single valve array inlet 146 and valve outlet 148.The valve inlet 146 and the valve outlet 148 are formed in the MSTchannel layer 100 and each have respective liquid sensors 174 providingfeedback to the CMOS circuitry 86. At the valve inlet 146, the flowbifurcates into the first flow-path 324 and the second flow-path 326,both of which extend between the valve inlet 146 and valve outlet 148.The first and second boiling-initiated valves (412 and 416) arepositioned along the first flow-path 324 and the third and fourthboiling-initiated valves (414 and 418) are on the second flow-path 326.

Liquid sensors 174 are also positioned between the two boiling-initiatedvalves in each flow-path. The sensors register which, if any, of thefirst, second, third or fourth boiling-initiated valves fail. If thefirst or the third boiling-initiated valves (412 and 414) fail, thesample flow is most likely restrained by the second and fourthboiling-initiated valves (416 and 418). The fault tolerant multiplevalve array 448 only fails in the unlikely event both boiling-initiatedvalves in the first flow-path 324 or second flow-paths 326 fail. Forgreater fault tolerance, the fault tolerant multiple valve array 448 isarbitrarily expandable in terms of the number of fluid flow-paths andthe number of boiling-initiated valves on each flow-path.

If the valve array operates without any boiling-initiated valve failure,the flow from the valve inlet 146 pins a meniscus in valve uptakes 428and 430 in the first boiling-initiated valve 412 and the thirdboiling-initiated valve 414 respectively. To open the boiling-initiatedvalves 412 and 414, the CMOS circuitry 86 sends activation pulses to theboiling-initiated valve heater contacts 153 in both valves. The heater152 in both boiling-initiated valves 412 and 414 heats the liquid at themeniscus above boiling temperature. Boiling unpins the menisci at bothvalve uptakes 428 and 430 such that capillary driven flow fills the capchannels 420 and 422 respectively.

The valve downtakes 432 and 434 are configured so as not to pin ameniscus and arrest the capillary driven flow. The flow into the capchannels 420 and 422 continues into the first flow-path MST channel 424and the second flow-path MST channel 426 via the valve downtakes 432 and434 respectively.

Capillary driven flow continues to the second and fourthboiling-initiated valves 416 and 418. Once again the flow pins ameniscus at the valve uptakes 436 and 438 of each boiling-initiatedvalve respectively. The liquid sensors 174 in each of the MST channels424 and 426 provide feedback to the CMOS circuitry 86 which times theactivation pulses for the valve heaters 152 accordingly. As with thefirst and third boiling-initiated valves 412 and 414, activating theheaters 152 in the second and fourth boiling-initiated valves 416 and418 boils liquid at the meniscus to unpin it from the uptakes 436 and438. Capillary driven flow into the cap channels 440 and 442 is followedby capillary driven flow through the downtakes 444 and 446 respectively.Flow along the first flow-path 324 reunites with flow in the secondflow-path 326 at the valve outlet 148 where the liquid sensor 174signals the CMOS circuitry 86 that the fault tolerant multiple valvearray 448 has opened.

Electroexplosive Valve

FIG. 122 is a plan view of an electroexplosive valve 71. The sampleflows along the MST channel 90 and into the valve inlet 146 formed bythe valve uptake 151. The liquid sample 119 fills the cap valveinterface cavity 73, stopping one side of the roof layer membrane 75when a meniscus 120 pins to each aperture 306. In this state, the valveis closed. When the CMOS circuitry 86 forces current through theresistors 77, the point along each resistor of highest current density79 explodes to destroy a roof layer rigid link 81 connecting themembrane 75 to fixed surroundings. With the membrane 75 unsupported anddislodged, the valve is open, and the liquid sample 119 flows from thevalve inlet 146 to the valve outlet 148.

Thermal-Bend-Actuated Bend-and-Break Valve

A thermal-bend-actuated bend-and-break valve 83 is shown in plan view inFIG. 123. The sample flows along the MST channel 90 and into the valveinlet 146 formed by the valve uptake 151. The liquid sample 119 fillsthe cap valve interface cavity 73, stopping one side of the roof layercantilevered section 162 and connected membrane 75 when a meniscus 120pins at each aperture 306. In this state, the valve is closed. A seriesof electrical pulses from the CMOS circuitry 86 to the thermal bendactuator 304 forces the roof layer cantilevered section 162, to whichthe thermal bend actuator 304 is bonded, to deflect towards thepassivation layer 88 by differential thermal expansion, breaking theweak roof layer rigid links 81 connecting the membrane 75 to itssurroundings. With the membrane 75 unsupported and dislodged, the valveis open, and the liquid sample 119 flows from the valve inlet 146 to thevalve outlet 148.

Dual Thermal-Bend-Actuated Bend-and-Break Valve

A dual thermal-bend-actuated bend-and-break valve 85 is shown in planview in FIG. 124. The sample flows along the MST channel 90 and into thevalve inlet 146 formed by the valve uptake 151. The liquid sample 119fills the cap valve interface cavity 73, stopping one side of both rooflayer cantilevered sections 162 and the connected membrane 75 when ameniscus 120 pins at each aperture 306. In this state, the valve isclosed. Both thermal bend actuators 304 are bonded to a roof layercantilevered section 162 and deflect towards the passivation layer 88 bydifferential thermal expansion upon receiving an electrical pulse fromthe CMOS circuitry 86 to break the rigid links 81 connecting themembrane 75 to its surroundings. With the membrane 75 unsupported anddislodged, the valve is open, and the liquid sample 119 flows from thevalve inlet 146 to the valve outlet 148.

Stiction Valve

A stiction valve 89 is shown in plan view in FIG. 125. The sample flowsalong the MST channel 90 and into the valve inlet 146 formed by thevalve uptake 151. The liquid sample 119 fills the cap valve interfacecavity 73, stopping one side of the roof layer cantilevered section 162and connected membrane 75 when a meniscus 120 pins at each aperture 306.In this state, the valve is closed. A series of electrical pulses fromthe CMOS circuitry 86 to the thermal bend actuator 304 forces the rooflayer cantilevered section 162, to which the thermal bend actuator 304is bonded, to deflect by differential thermal expansion, forcing themembrane 75 into contact with the passivation layer 88. With stictionbetween the membrane 75 and passivation layer 88 counterbalancing therestorative force, and deformation of the flexible link 91 accommodatingthe cantilevered section 162 restorative displacement, the valve remainsstatically open for the liquid sample 119 to flow from the valve inlet146 to the valve outlet 148.

Stiction Valve Variant

A stiction valve variant 93 is shown in plan view in FIG. 126. Thesample flows along the MST channel 90 and into the valve inlet 146formed by the valve uptake 151. The liquid sample 119 fills the capvalve interface cavity 73, stopping one side of the roof layercantilevered section 162 when a meniscus 120 pins at each aperture 306.In this state, the valve is closed. A series of electrical pulses fromthe CMOS circuitry 86 to the thermal bend actuator 304 forces the rooflayer cantilevered section 162, to which the thermal bend actuator 304is bonded, to deflect by differential thermal expansion and contact thepassivation layer 88. With stiction between the cantilevered section 162and passivation layer 88 counterbalancing the restorative force, and thelength of the cantilevered section 162 sufficient so that its mechanicalflexibility accommodates restorative displacement of the thermal bendactuator 304, the valve remains statically open for the liquid sample119 to flow from the valve inlet 146 to the valve outlet 148.

Bubble Break Valve

A bubble break valve 95 is shown in plan view in FIG. 127. The sampleflows along the MST channel 90 and into the valve inlet 146 formed bythe valve uptake 151. The liquid sample 119 fills the cap valveinterface cavity 73, stopping one side of the roof layer membrane 75when the meniscus 120 pins at the aperture 306. In this state, the valveis closed. The annular heater 152 is resistively heated by an electricalpulse from the CMOS circuitry 86 until a vapor bubble is generated inthe liquid sample that forces the membrane 75 to rotate around and breakthe roof layer rigid link 81. With the membrane 75 unsupported anddislodged, the valve is open, and the liquid sample 119 flows from thevalve inlet 146 to the valve outlet 148.

Other Valve Array Variants

Any of the valve variants described above can be used to form a valvearray. Furthermore, the valve array can include different types ofvalve.

Dialysis Variants Leukocyte Target

The dialysis design described above in the LOC device 301 targetspathogens. FIG. 72 is a schematic section view of a dialysis section 328designed to concentrate leukocytes from a blood sample for human DNAanalysis. It will be appreciated that the structure is essentially thesame as that of the pathogen target dialysis section 70 described abovewith the exception that apertures in the form of 7.5 micron diameterholes 165 restrict leukocytes from passing from the cap channel 94 tothe dialysis MST channels 204. In situations where the sample beinganalysed is a blood sample, and the presence of haemoglobin from theerythrocytes interferes with the subsequent reaction steps, addition ofan erythrocyte lysis buffer along with the anticoagulant in thereservoir 54 (see FIG. 22), will ensure that the majority of the lysederythrocytes (and hence haemoglobin) will be removed from the sampleduring this dialysis step. A commonly used erythrocyte lysis buffer is0.15M NH₄CL, 10 mM KHCO₃, 0.1 mM EDTA, pH 7.2-7.4, but a person skilledin the art will recognise that any buffer which efficiently lysederythrocytes can be used.

Downstream of the leukocyte dialysis section 328, the cap channel 94becomes the target channel 74 such that the leukocytes continue as partof the assay. Furthermore, in this case, the dialysis uptake holes 168lead to a waste channel 72 so that all smaller cells and components inthe sample are removed. It should be noted that this dialysis variantonly reduces the concentration of the unwanted specimens in the targetchannel 74.

FIG. 100 schematically illustrates a large constituents dialysis section686 which also separates any large target constituents from a sample.The apertures in this dialysis section are fabricated with a size andshape tailored to withhold the large target constituents of interest inthe target channel for further analysis. As with the leukocyte dialysissection described above, most (but not all) smaller sized cells,organisms or molecules flow to a waste reservoir 768. Thus, otherembodiments of the LOC device are not limited to separating leukocytesthat are larger than 7.5 μm in size, but can be used to separate cells,organisms or molecules of any size desired.

Nucleic Acid Amplification Variants Direct PCR

Traditionally, PCR requires extensive purification of the target DNAprior to preparation of the reaction mixture. However, with appropriatechanges to the chemistry and sample concentration, it is possible toperform nucleic acid amplification with minimal DNA purification, ordirect amplification. When the nucleic acid amplification process isPCR, this approach is called direct PCR. In LOC devices where nucleicacid amplification is performed at a controlled, constant temperature,the approach is direct isothermal amplification. Direct nucleic acidamplification techniques have considerable advantages for use in LOCdevices, particularly relating to simplification of the required fluidicdesign. Adjustments to the amplification chemistry for direct PCR ordirect isothermal amplification include increased buffer strength, theuse of polymerases which have high activity and processivity, andadditives which chelate with potential polymerase inhibitors. Dilutionof inhibitors present in the sample is also important.

To take advantage of direct nucleic acid amplification techniques, theLOC device designs incorporate two additional features. The firstfeature is reagent reservoirs (for example reservoir 58 in FIG. 8) whichare appropriately dimensioned to supply a sufficient quantity ofamplification reaction mix, or diluent, so that the final concentrationsof sample components which might interfere with amplification chemistryare low enough to permit successful nucleic acid amplification. Thedesired dilution of non-cellular sample components is in the range of 5×to 20×. Different LOC structures, for example the pathogen dialysissection 70 in FIG. 4, are used when appropriate to ensure that theconcentration of target nucleic acid sequences is maintained at a highenough level for amplification and detection. In this embodiment,further illustrated in FIG. 6, a dialysis section which effectivelyconcentrates pathogens small enough to be passed into the amplificationsection 292 is employed upstream of the sample extraction section 290,and rejects larger cells to a waste receptacle 76. In anotherembodiment, a dialysis section is used to selectively deplete proteinsand salts in blood plasma while retaining cells of interest.

The second LOC structural feature which supports direct nucleic acidamplification is design of channel aspect ratios to adjust the mixingratio between the sample and the amplification mix components. Forexample, to ensure dilution of inhibitors associated with the sample inthe preferred 5×-20× range through a single mixing step, the length andcross-section of the sample and reagent channels are designed such thatthe sample channel, upstream of the location where mixing is initiated,constitutes a flow impedance 4×-19× higher than the flow impedance ofthe channels through which the reagent mixture flows. Control over flowimpedances in microchannels is readily achieved through control over thedesign geometry. The flow impedance of a microchannel increases linearlywith the channel length, for a constant cross-section. Importantly formixing designs, flow impedance in microchannels depends more strongly onthe smallest cross-sectional dimension. For example, the flow impedanceof a microchannel with rectangular cross-section is inverselyproportional to the cube of the smallest perpendicular dimension, whenthe aspect ratio is far from unity.

Reverse-Transcriptase PCR (RT-PCR)

Where the sample nucleic acid species being analysed or extracted isRNA, such as from RNA viruses or messenger RNA, it is first necessary toreverse transcribe the RNA into complementary DNA (cDNA) prior to PCRamplification. The reverse transcription reaction can be performed inthe same chamber as the PCR (one-step RT-PCR) or it can be performed asa separate, initial reaction (two-step RT-PCR). In the LOC variantsdescribed herein, a one-step RT-PCR can be performed simply by addingthe reverse transcriptase to reagent reservoir 62 along with thepolymerase and programming the heaters 154 to cycle firstly for thereverse transcription step and then progress onto the nucleic acidamplification step. A two-step RT-PCR could also be easily achieved byutilizing the reagent reservoir 58 to store and dispense the buffers,primers, dNTPs and reverse transcriptase and the incubation section 114for the reverse transcription step followed by amplification in thenormal way in the amplification section 112.

Isothermal Nucleic Acid Amplification

For some applications, isothermal nucleic acid amplification is thepreferred method of nucleic acid amplification, thus avoiding the needto repetitively cycle the reaction components through varioustemperature cycles but instead maintaining the amplification section ata constant temperature, typically around 37° C. to 41° C. A number ofisothermal nucleic acid amplification methods have been described,including Strand Displacement Amplification (SDA), TranscriptionMediated Amplification (TMA), Nucleic Acid Sequence Based Amplification(NASBA), Recombinase Polymerase Amplification (RPA), Helicase-Dependentisothermal DNA Amplification (HDA), Rolling Circle Amplification (RCA),Ramification Amplification (RAM) and Loop-mediated IsothermalAmplification (LAMP), and any of these, or other isothermalamplification methods, can be employed in particular embodiments of theLOC device described herein.

In order to perform isothermal nucleic acid amplification, the reagentreservoirs 60 and 62 adjoining the amplification section will be loadedwith the appropriate reagents for the specified isothermal methodinstead of PCR amplification mix and polymerase. For example, for SDA,reagent reservoir 60 contains amplification buffer, primers and dNTPsand reagent reservoir 62 contains an appropriate nickase enzyme andExo-DNA polymerase. For RPA, reagent reservoir 60 contains theamplification buffer, primers, dNTPs and recombinase proteins, withreagent reservoir 62 containing a strand displacing DNA polymerase suchas Bsu. Similarly, for HDA, reagent reservoir 60 contains amplificationbuffer, primers and dNTPs and reagent reservoir 62 contains anappropriate DNA polymerase and a helicase enzyme to unwind the doublestranded DNA strand instead of using heat. The skilled person willappreciate that the necessary reagents can be split between the tworeagent reservoirs in any manner appropriate for the nucleic acidamplification process.

For amplification of viral nucleic acids from RNA viruses such as HIV orhepatitis C virus, NASBA or TMA is appropriate as it is unnecessary tofirst transcribe the RNA to cDNA. In this example, reagent reservoir 60is filled with amplification buffer, primers and dNTPs and reagentreservoir 62 is filled with RNA polymerase, reverse transcriptase and,optionally, RNase H.

For some forms of isothermal nucleic acid amplification it may benecessary to have an initial denaturation cycle to separate the doublestranded DNA template, prior to maintaining the temperature for theisothermal nucleic acid amplification to proceed. This is readilyachievable in all embodiments of the LOC device described herein, as thetemperature of the mix in the amplification section 112 can be carefullycontrolled by the heaters 154 in the amplification microchannels 158(see FIG. 14).

Isothermal nucleic acid amplification is more tolerant of potentialinhibitors in the sample and, as such, is generally suitable for usewhere direct nucleic acid amplification from the sample is desired.Therefore, isothermal nucleic acid amplification is sometimes useful inLOC variant XLIII 673, LOC variant XLIV 674 and LOC variant XLVII 677,amongst others, shown in FIGS. 101, 102 and 103, respectively. Directisothermal amplification may also be combined with one or morepre-amplification dialysis steps 70, 686 or 682 as shown in FIGS. 101and 103 and/or a pre-hybridization dialysis step 682 as indicated inFIG. 102 to help partially concentrate the target cells in the samplebefore nucleic acid amplification or remove unwanted cellular debrisprior to the sample entering the hybridization chamber array 110,respectively. The person skilled in the art will appreciate that anycombination of pre-amplification dialysis and pre-hybridization dialysiscan be used.

Isothermal nucleic acid amplification can also be performed in parallelamplification sections such as those schematically represented in FIGS.96, 97 and 98, multiplexed and some methods of isothermal nucleic acidamplification, such as LAMP, are compatible with an initial reversetranscription step to amplify RNA.

Additional Details on the Fluorescence Detection System

FIGS. 58 and 59 show the hybridization-responsive FRET probes 236. Theseare often referred to as molecular beacons and are stem-and-loop probes,generated from a single strand of nucleic acid, that fluoresce uponhybridization to complementary nucleic acids. FIG. 58 shows a singleFRET probe 236 prior to hybridization with a target nucleic acidsequence 238. The probe has a loop 240, stem 242, a fluorophore 246 atthe 5′ end, and a quencher 248 at the 3′ end. The loop 240 consists of asequence complementary to the target nucleic acid sequence 238.Complementary sequences on either side of the probe sequence annealtogether to form the stem 242.

In the absence of a complementary target sequence, the probe remainsclosed as shown in FIG. 58. The stem 242 keeps the fluorophore-quencherpair in close proximity to each other, such that significant resonantenergy transfer can occur between them, substantially eliminating theability of the fluorophore to fluoresce when illuminated with theexcitation light 244.

FIG. 59 shows the FRET probe 236 in an open or hybridized configuration.Upon hybridization to a complementary target nucleic acid sequence 238,the stem-and-loop structure is disrupted, the fluorophore and quencherare spatially separated, thus restoring the ability of the fluorophore246 to fluoresce. The fluorescence emission 250 is optically detected asan indication that the probe has hybridized.

The probes hybridize with very high specificity with complementarytargets, since the stem helix of the probe is designed to be more stablethan a probe-target helix with a single nucleotide that is notcomplementary. Since double-stranded DNA is relatively rigid, it issterically impossible for the probe-target helix and the stem helix tocoexist.

Primer-Linked Probes

Primer-linked, stem-and-loop probes and primer-linked, linear probes,otherwise known as scorpion probes, are an alternative to molecularbeacons and can be used for real-time and quantitative nucleic acidamplification in the LOC device. Real-time amplification could beperformed directly in the hybridization chambers of the LOC device. Thebenefit of using primer-linked probes is that the probe element isphysically linked to the primer, thus only requiring a singlehybridization event to occur during the nucleic acid amplificationrather than separate hybridizations of the primers and probes beingrequired. This ensures that the reaction is effectively instantaneousand results in stronger signals, shorter reaction times and betterdiscrimination than when using separate primers and probes. The probes(along with polymerase and the amplification mix) would be depositedinto the hybridization chambers 180 during fabrication and there wouldbe no need for a separate amplification section on the LOC device.Alternatively, the amplification section is left unused or used forother reactions.

Primer-Linked Linear Probe

FIGS. 104 and 105 show a primer-linked linear probe 692 during theinitial round of nucleic acid amplification and in its hybridizedconfiguration during subsequent rounds of nucleic acid amplification,respectively. Referring to FIG. 104, the primer-linked linear probe 692has a double-stranded stem segment 242. One of the strands incorporatesthe primer linked probe sequence 696 which is homologous to a region onthe target nucleic acid 696 and is labelled on its 5′ end withfluorophore 246, and linked on its 3′ end to an oligonucleotide primer700 via an amplification blocker 694. The other strand of the stem 242is labelled at its 3 end with a quencher moiety 248. After an initialround of nucleic acid amplification has completed, the probe can looparound and hybridize to the extended strand with the, now complementary,sequence 698. During the initial round of nucleic acid amplification,the oligonucleotide primer 700 anneals to the target DNA 238 (FIG. 104)and is then extended, forming a DNA strand containing both the probesequence and the amplification product. The amplification blocker 694prevents the polymerase from reading through and copying the proberegion 696. Upon subsequent denaturation, the extended oligonucleotideprimer 700/template hybrid is dissociated and so is the double strandedstem 242 of the primer-linked linear probe, thus releasing the quencher248. Once the temperature decreases for the annealing and extensionsteps, the primer linked probe sequence 696 of the primer-linked linearprobe curls around and hybridizes to the amplified complementarysequence 698 on the extended strand and fluorescence is detectedindicating the presence of the target DNA. Non-extended primer-linkedlinear probes retain their double-stranded stem and fluorescence remainsquenched. This detection method is particularly well suited for fastdetection systems as it relies on a single-molecule process.

Primer-Linked Stem-and-Loop Probes

FIGS. 106A to 106F show the operation of a primer-linked stem-and-loopprobe 704. Referring to FIG. 106A, the primer-linked stem-and-loop probe704 has a stem 242 of complementary double-stranded DNA and a loop 240which incorporates the probe sequence. One of the stem strands 708 islabelled at its 5′ end with fluorophore 246. The other strand 710 islabelled with a 3′-end quencher 248 and carries both the amplificationblocker 694 and oligonucleotide primer 700. During the initialdenaturation phase (see FIG. 106B), the strands of the target nucleicacid 238 separate, as does the stem 242 of the primer-linked,stem-and-loop probe 704. When the temperature cools for the annealingphase (see FIG. 106C), the oligonucleotide primer 700 on theprimer-linked stem-and-loop probe 704 hybridizes to the target nucleicacid sequence 238. During extension (see FIG. 106D) the complement 706to the target nucleic acid sequence 238 is synthesized forming a DNAstrand containing both the probe sequence 704 and the amplified product.The amplification blocker 694 prevents the polymerase from readingthrough and copying the probe region 704. When the probe next anneals,following denaturation, the probe sequence of the loop segment 240 ofthe primer-linked stem-and-loop probe (see FIG. 106F) anneals to thecomplementary sequence 706 on the extended strand. This configurationleaves the fluorophore 246 relatively remote from the quencher 248,resulting in a significant increase in fluorescence emission.

Control Probes

The hybridization chamber array 110 includes some hybridization chambers180 with positive and negative control probes used for assay qualitycontrol. FIGS. 118 and 119 schematically illustrate negative controlprobes without a fluorophore 796, and FIGS. 120 and 121 are sketches ofpositive control probes without a quencher 798. The positive andnegative control probes have a stem-and-loop structure like the FRETprobes described above. However, a fluorescence signal 250 will alwaysbe emitted from positive control probes 798 and no fluorescence signal250 is ever emitted from negative control probes 796, regardless ofwhether the probes hybridize into an open configuration or remainclosed.

Referring to FIGS. 118 and 119, the negative control probe 796 has nofluorophore (and may or may not have a quencher 248). Hence, whether thetarget nucleic acid sequence 238 hybridizes with the probe (see FIG.119), or the probe remains in its stem-and-loop configuration (see FIG.118), the response to the excitation light 244 is negligible.Alternatively, the negative control probe 796 could be designed so thatit always remains quenched. For example, by synthesizing the loop 240 tohave a probe sequence that will not hybridize to any nucleic acidsequence within the sample under investigation, the stem 242 of theprobe molecule will re-hybridize to itself and the fluorophore andquencher will remain in close proximity and no appreciable fluorescencesignal will be emitted. This negative control signal would correspond tolow level emissions from hybridization chambers 180 in which the probeshas not hybridized but the quencher does not quench all emissions fromthe reporter.

Conversely, the positive control probe 798 is constructed without aquencher as illustrated in FIGS. 120 and 121. Nothing quenches thefluorescence emission 250 from the fluorophore 246 in response to theexcitation light 244 regardless of whether the positive control probe798 hybridizes with the target nucleic acid sequence 238.

FIG. 52 shows a possible distribution of the positive and negativecontrol probes (378 and 380 respectively) throughout the hybridizationchamber array 110. The control probes 378 and 380 are placed inhybridization chambers 180 positioned in a line across the hybridizationchamber array 110. However, the arrangement of the control probes withinthe array is arbitrary (as is the configuration of the hybridizationchamber array 110).

Fluorophore Design

Fluorophores with long fluorescence lifetimes are required in order toallow enough time for the excitation light to decay to an intensitybelow that of the fluorescence emission at which time the photosensor 44is enabled, thereby providing a sufficient signal to noise ratio. Also,longer fluorescence lifetime translates into larger integratedfluorescence photon count.

The fluorophores 246 (see FIG. 59) have a fluorescence lifetime greaterthan 100 nanoseconds, often greater than 200 nanoseconds, more commonlygreater than 300 nanoseconds and in most cases greater than 400nanoseconds.

The metal-ligand complexes based on the transition metals or lanthanideshave long lifetimes (from hundreds of nanoseconds to milliseconds),adequate quantum yields, and high thermal, chemical and photochemicalstability, which are all favourable properties with respect to thefluorescence detection system requirements.

A particularly well-studied metal-ligand complex based on the transitionmetal ion Ruthenium (Ru (II)) is tris(2,2′-bipyridine) ruthenium (II)([Ru(bpy)₃]²⁺) which has a lifetime of approximately 1 μs. This complexis available commercially from Biosearch Technologies under the brandname Pulsar 650.

TABLE 1 Photophysical properties of Pulsar 650 (Ruthenium chelate)Parameter Symbol Value Unit Absorption Wavelength λ_(abs) 460 nmEmission Wavelength λ_(em) 650 nm Extinction Coefficient E 14800 M⁻¹cm⁻¹Fluorescence Lifetime τ_(f) 1.0 μs Quantum Yield H 1 (deoxygenated) N/A

Terbium chelate, a lanthanide metal-ligand complex has been successfullydemonstrated as a fluorescent reporter in a FRET probe system, and alsohas a long lifetime of 1600 μs.

TABLE 2 Photophysical properties of terbium chelate Parameter SymbolValue Unit Absorption Wavelength λ_(abs) 330-350 nm Emission Wavelengthλ_(em) 548 nm Extinction Coefficient E 13800 M⁻¹cm⁻¹ (λ_(abs) and liganddepen- dent, can be up to 30000 @ λ_(e = 340 nm)) Fluorescence Lifetimeτ_(f) 1600 μs (hybridized probe) Quantum Yield H 1 N/A (liganddependent)

The fluorescence detection system used by the LOC device 301 does notutilize filters to remove unwanted background fluorescence. It istherefore advantageous if the quencher 248 has no native emission inorder to increase the signal-to-noise ratio. With no native emission,there is no contribution to background fluorescence from the quencher248. High quenching efficiency is also important so that fluorescence isprevented until a hybridization event occurs. The Black Hole Quenchers(BHQ), available from Biosearch Technologies, Inc. of Novato Calif.,have no native emission and high quenching efficiency, and are suitablequenchers for the system. BHQ-1 has an absorption maximum at 534 nm, anda quenching range of 480-580 nm, making it a suitable quencher for theTb-chelate fluorophore. BHQ-2 has an absorption maximum at 579 nm, and aquenching range of 560-670 nm, making it a suitable quencher for Pulsar650.

Iowa Black Quenchers (Iowa Black FQ and RQ), available from IntegratedDNA Technologies of Coralville, Iowa, are suitable alternative quencherswith little or no background emission. Iowa Black FQ has a quenchingrange from 420-620 nm, with an absorption maximum at 531 nm and wouldtherefore be a suitable quencher for the Tb-chelate fluorophore. IowaBlack RQ has an absorption maximum at 656 nm, and a quenching range of500-700 nm, making it an ideal quencher for Pulsar 650.

In the embodiments described here, the quencher 248 is a functionalmoiety which is initially attached to the probe, but other embodimentsare possible in which the quencher is a separate molecule free insolution.

Excitation Source

In the fluorescence detection based embodiments described herein, a LEDis chosen as the excitation source instead of a laser diode, high powerlamp or laser due to the low power consumption, low cost and small size.Referring to FIG. 107, the LED 26 is positioned directly above thehybridization chamber array 110 on an external surface of the LOC device301. On the opposing side of the hybridization chamber array 110, is thephotosensor 44, made up of an array of photodiodes 184 (see FIGS. 53, 54and 73) for detection of fluorescence signals from each of the chambers.

FIGS. 108, 109 and 110 schematically illustrate other embodiments forexposing the probes to excitation light. In the LOC device 30 shown inFIG. 108, the excitation light 244 generated by the excitation LED 26 isdirected onto the hybridization chamber array 110 by the lens 254. Theexcitation LED 26 is pulsed and the fluorescence emissions are detectedby the photosensor 44.

In the LOC device 30 shown in FIG. 109, the excitation light 244generated by the excitation LED 26 is directed onto the hybridizationchamber array 110 by the lens 254, a first optical prism 712 and secondoptical prism 714. The excitation LED 26 is pulsed and the fluorescenceemissions are detected by the photosensor 44.

Similarly, the LOC device 30 shown in FIG. 110, the excitation light 244generated by the excitation LED 26 is directed onto the hybridizationchamber array 110 by the lens 254, a first minor 716 and second minor718. Again, the excitation LED 26 is pulsed and the fluorescenceemissions are detected by the photosensor 44.

The excitation wavelength of the LED 26 is dependent on the choice offluorescent dye. The Philips LXK2-PR14-R00 is a suitable excitationsource for the Pulsar 650 dye. The SET UVTOP335TO39BL LED is a suitableexcitation source for the Tb-chelate label.

TABLE 3 Philips LXK2-PR14-R00 LED specifications Parameter Symbol ValueUnit Wavelength λ_(ex) 460 nm Emission Frequency ν_(em) 6.52(10)¹⁴ HzOutput Power p_(l) 0.515 (min) @ 1 A W Radiation pattern Lambertianprofile N/A

TABLE 4 SET UVTOP334TO39BL LED Specifications Parameter Symbol ValueUnit Wavelength λ_(e) 340 nm Emission Frequency ν_(e) 8.82(10)¹⁴ HzPower p_(l) 0.000240 (min) @ 20 mA W Pulse Forward Current I 200 mARadiation pattern Lambertian N/A

Ultra Violet Excitation Light

Silicon absorbs little light in the UV spectrum. Accordingly, it isadvantageous to use

UV excitation light. A UV LED excitation source can be used but thebroad spectrum of the LED 26 reduces the effectiveness of this method.To address this, a filtered UV LED can be used. Optionally, a UV lasercan be the excitation source unless the relatively high cost of thelaser is impractical for the particular test module market.

LED Driver

The LED driver 29 drives the LED 26 at a constant current for therequired duration. A lower power USB 2.0-certifiable device can draw atmost 1 unit load (100 mA), with a minimum operating voltage of 4.4 V. Astandard power conditioning circuit is used for this purpose.

Photodiode

FIG. 54 shows the photodiode 184 integrated into the CMOS circuitry 86of the LOC device 301. The photodiode 184 is fabricated as part of theCMOS circuitry 86 without additional masks or steps. This is onesignificant advantage of a CMOS photodiode over a CCD, an alternatesensing technology which could be integrated on the same chip usingnon-standard processing steps, or fabricated on an adjacent chip.On-chip detection is low cost and reduces the size of the assay system.The shorter optical path length reduces noise from the surroundingenvironment for efficient collection of the fluorescence signal andeliminates the need for a conventional optical assembly of lenses andfilters.

Quantum efficiency of the photodiode 184 is the fraction of photonsimpinging on its active area 185 that are effectively converted tophoto-electrons. For standard silicon processes, the quantum efficiencyis in the range of 0.3 to 0.5 for visible light, depending on processparameters such as the amount and absorption properties of the coverlayers.

The detection threshold of the photodiode 184 determines the smallestintensity of the fluorescence signal that can be detected. The detectionthreshold also determines the size of the photodiode 184 and hence thenumber of hybridization chambers 180 in the hybridization and detectionsection 52 (see FIG. 52). The size and number of chambers are technicalparameters that are limited by the dimensions of the LOC device (in thecase of the LOC device 301, the dimensions are 1760 μm×5824 μm) and thereal estate available after other functional modules such as thepathogen dialysis section 70 and amplification section(s) 112 areincorporated.

For standard silicon processes, the photodiode 184 detects a minimum of5 photons. However, to ensure reliable detection, the minimum can be setto 10 photons. Therefore with the quantum efficiency range being 0.3 to0.5 (as discussed above), the fluorescence emission from the probesshould be a minimum of 17 photons but 30 photons would incorporate asuitable margin of error for reliable detection.

Calibration Chambers

The non-uniformity of the electrical characteristic of the photodiode184, autofluorescence, and residual excitation photon flux that has notyet completely decayed, introduce background noise and offset into theoutput signal. This background is removed from each output signal usingone or more calibration signals. Calibration signals are generated byexposing one or more calibration photodiodes 184 in the array torespective calibration sources. A low calibration source is used fordetermining a negative result in which a target has not reacted with aprobe. A high calibration source is indicative of a positive result froma probe-target complex. In the embodiment described here, the lowcalibration light source is provided by calibration chambers 382 in thehybridization chamber array 110 which:

do not contain any probes;

contain probes that have no fluorescent reporter; or,

contain probes with a reporter and quencher configured such thatquenching is always expected to occur.

The output signal from such calibration chambers 382 closelyapproximates the noise and offset in the output signal from all thehybridization chambers in the LOC device. Subtracting the calibrationsignal from the output signals generated by the other hybridizationchambers substantially removes the background and leaves the signalgenerated by the fluorescence emission (if any). Signals arising fromambient light in the region of the chamber array are also subtracted.

It will be appreciated that the negative control probes described abovewith reference to FIGS. 118 to 121 can be used in calibration chambers.However, as shown in FIGS. 112 and 113, which are enlarged views ofinsets DG and DH of LOC variant X 728 shown in FIG. 111, another optionis to fluidically isolate the calibration chambers 382 from theamplicon. The background noise and offset can be determined by leavingthe fluidically isolated chambers empty, or containing reporterlessprobes, or indeed any of the ‘normal’ probes with both reporter andquencher as hybridization is precluded by fluidic isolation.

The calibration chambers 382 can provide a high calibration source togenerate a high signal in the corresponding photodiodes. The high signalcorresponds to all probes in a chamber having hybridized. Spottingprobes with reporters and no quenchers, or just reporters willconsistently provide a signal approximating that of a hybridizationchamber in which a predominant number of the probes have hybridized. Itwill also be appreciated that calibration chambers 382 can be usedinstead of control probes, or in addition to control probes.

The number and arrangement of the calibration chambers 382 throughoutthe hybridization chamber array is arbitrary. However, the calibrationis more accurate if photodiodes 184 are calibrated by a calibrationchamber 382 that is relatively proximate. Referring to FIG. 56, thehybridization chamber array 110 has one calibration chamber 382 forevery eight hybridization chambers 180. That is, a calibration chamber382 is positioned in the middle of every three by three square ofhybridization chambers 180. In this configuration, the hybridizationchambers 180 are calibrated by a calibration chamber 382 that isimmediately adjacent.

FIG. 117 shows a differential imager circuit 788 used to subtract thesignal from the photodiode 184 corresponding to the calibration chamber382 as a result of excitation light, from the fluorescence signal fromthe surrounding hybridization chambers 180. The differential imagercircuit 788 samples the signal from the pixel 790 and a “dummy” pixel792. In one embodiment, the “dummy” pixel 792 is shielded from light, soits output signal provides a dark reference. Alternatively, the “dummy”pixel 792 can be exposed to the excitation light along with the rest ofthe array. In the embodiment where the “dummy” pixel 792 is open tolight, signals arising from ambient light in the region of the chamberarray are also subtracted. The signals from the pixel 790 are small(i.e. close to dark signal), and without a reference to a dark level itis hard to differentiate between the background and a very small signal.

During use, the “read_row” 794 and “read_row_d” 795 are activated and M4797 and MD4 801 transistors are turned on. Switches 807 and 809 areclosed such that the outputs from the pixel 790 and “dummy” pixel 792are stored on pixel capacitor 803 and dummy pixel capacitor 805respectively. After the pixel signals have been stored, switches 807 and809 are deactivated. Then the “read_col” switch 811 and dummy “read_col”switch 813 are closed, and the switched capacitor amplifier 815 at theoutput amplifies the differential signal 817.

Suppression and Enablement of the Photodiode

The photodiode 184 needs to be suppressed during excitation by the LED26 and enabled during fluorescence. FIG. 74 is a circuit diagram for asingle photodiode 184 and FIG. 75 is a timing diagram for the photodiodecontrol signals. The circuit has photodiode 184 and six MOS transistors,M_(shunt) 394, M_(tx) 396, M_(reset) 398, M_(sf) 400, M_(read) 402 andM_(bias) 404. At the beginning of the excitation cycle, t1, thetransistors M_(shunt) 394, and M_(reset) 398 are turned on by pullingthe M_(shunt) gate 384 and the reset gate 388 high. During this period,the excitation photons generate carriers in the photodiode 184. Thesecarriers have to be removed, as the amount of generated carriers can besufficient to saturate the photodiode 184. During this cycle, M_(shunt)394 directly removes the carriers generated in photodiode 184, whileM_(reset) 398 resets any carriers that have accumulated on node ‘NS’ 406due to leakage in transistors or due to diffusion of excitation-producedcarriers in the substrate. After excitation, a capture cycle commencesat t4. During this cycle, the emitted response from the fluorophore iscaptured and integrated in the circuit on node ‘NS’ 406. This isachieved by pulling tx gate 386 high, which turns on the transistorM_(tx) 396 and transfers any accumulated carriers on the photodiode 184to node ‘NS’ 406. The duration of the capture cycle can be as long asthe fluorophore emits. The outputs from all photodiodes 184 in thehybridization chamber array 110 are captured simultaneously.

There is a delay between the end of the capture cycle t5 and the startof the read cycle t6. This delay is due to the requirement to read eachphotodiode 184 in the hybridization chamber array 110 (see FIG. 52)separately following the capture cycle. The first photodiode 184 to beread will have the shortest delay before the read cycle, while the lastphotodiode 184 will have the longest delay before the read cycle. Duringthe read cycle, transistor M_(read) 402 is turned on by pulling the readgate 393 high. The ‘NS’ node 406 voltage is buffered and read out usingthe source-follower transistor M_(sf) 400.

There are additional, optional methods of enabling or suppressing thephotodiode as discussed below:

1. Suppression Methods

FIGS. 114, 115 and 116 show three possible configurations 778, 780, 782for the M_(shunt) transistor 394. The M_(shunt) transistor 394 has avery high off ratio at maximum |V_(GS)|=5 V which is enabled duringexcitation. As shown in FIG. 114, the M_(shunt) gate 384 is configuredto be on the edge of the photodiode 184. Optionally, as shown in FIG.115, the M_(shunt) gate 384 may be configured to surround the photodiode184. A third option is to configure the M_(shunt) gate 384 inside thephotodiode 184, as shown in FIG. 116. Under this third option therewould be less photodiode active area 185.

These three configurations 778, 780 and 782 reduce the average pathlength from all locations in the photodiode 184 to the M_(shunt) gate384. In FIG. 114, the M_(shunt) gate 384 is on one side of thephotodiode 184. This configuration is simplest to fabricate and impingesthe least on the photodiode active area 185. However, any carrierslingering on the remote side of the photodiode 184 would take longer topropagate through to the M_(shunt) gate 384.

In FIG. 115, the M_(shunt) gate 384 surrounds the photodiode 184. Thisfurther reduces the average path length for carriers in the photodiode184 to the M_(shunt) gate 384. However, extending the M_(shunt) gate 384about the periphery of the photodiode 184 imposes a greater reduction ofthe photodiode active area 185. The configuration 782 in FIG. 116positions the M_(shunt) gate 384 within the active area 185. Thisprovides the shortest average path length to the M_(shunt) gate 384 andhence the shortest transition time. However, the impingement on theactive area 185 is greatest. It also poses a wider leakage path.

2. Enabling Methods

a. A trigger photodiode drives the shunt transistor with a fixed delay.b. A trigger photodiode drives the shunt transistor with programmabledelay.c. The shunt transistor is driven from the LED drive pulse with a fixeddelay.d. The shunt transistor is driven as in 2c but with programmable delay.

FIG. 77 is a schematic section view through a hybridization chamber 180showing a photodiode 184 and trigger photodiode 187 embedded in the CMOScircuitry 86. A small area in the corner of the photodiode 184 isreplaced with the trigger photodiode 187. A trigger photodiode 187 witha small area is sufficient as the intensity of the excitation light willbe high in comparison with the fluorescence emission. The triggerphotodiode 187 is sensitive to the excitation light 244. The triggerphotodiode 187 registers that the excitation light 244 has extinguishedand activates the photodiode 184 after a short time delay Δt 300 (seeFIG. 2). This delay allows the fluorescence photodiode 184 to detect thefluorescence emission from the FRET probes 186 in the absence of theexcitation light 244. This enables detection and improves the signal tonoise ratio.

Both photodiodes 184 and trigger photodiodes 187 are located in the CMOScircuitry 86 under each hybridization chamber 180. The array ofphotodiodes combines, along with appropriate electronics, to form thephotosensor 44 (see FIG. 73). The photodiodes 184 are pn-junctionfabricated during CMOS structure manufacturing without additional masksor steps. During MST fabrication, the dielectric layer (not shown) abovethe photodiodes 184 is optionally thinned using the standard MSTphotolithography techniques to allow more fluorescent light toilluminate the active area 185 of the photodiode 184. The photodiode 184has a field of view such that the fluorescence signal from theprobe-target hybrids within the hybridization chamber 180 is incident onthe sensor face. The fluorescent light is converted into a photocurrentwhich can then be measured using CMOS circuitry 86.

Alternatively, one or more hybridization chambers 180 can be dedicatedto a trigger photodiode 187 only. These options can be used in these incombination with 2a and 2b above.

Delayed Detection of Fluorescence

The following derivations elucidate the delayed detection offluorescence using a long-lifetime fluorophore for the LED/fluorophorecombinations described above. The fluorescence intensity is derived as afunction of time after excitation by an ideal pulse of constantintensity I, between time t₁ and t₂ as shown in FIG. 60.

Let [S1](t) equal the density of excited states at time t, then duringand after excitation, the number of excited states per unit time perunit volume is described by the following differential equation:

$\begin{matrix}{{{\frac{\left\lbrack {S\; 1} \right\rbrack}{t}(t)} + \frac{\left\lbrack {S\; 1} \right\rbrack (t)}{\tau_{F}}} = \frac{I_{e}ɛ\; c}{h\; \nu_{e}}} & (1)\end{matrix}$

where c is the molar concentration of fluorophores, ε is the molarextinction coefficient, ν_(e) is the excitation frequency, andh=6.62606896(10)⁻³⁴ Js is the Planck constant.This differential equation has the general form:

${\frac{y}{x} + {{p(x)}y}} = {q(x)}$

which has the solution:

$\begin{matrix}{{y(x)} = \frac{{\int{^{\int{{p{(x)}}{x}}}{q(x)}{x}}} + k}{^{\int{{p{(x)}}{x}}}}} & (2)\end{matrix}$

Using this now to solve equation (1),

$\begin{matrix}{{\left\lbrack {S\; 1} \right\rbrack (t)} = {\frac{I_{e}ɛ\; c\; \tau_{f}}{h\; \nu_{e}} + {k\; ^{{- t}/\tau_{f}}}}} & (3)\end{matrix}$

Now at time t₁, [S1](t₁)=0, and from (3):

$\begin{matrix}{k = {{- \frac{I_{e}ɛ\; c\; \tau_{f}}{h\; \nu_{e}}}^{t_{1}/\tau_{f}}}} & (4)\end{matrix}$

Substituting (4) into (3):

${\left\lbrack {S\; 1} \right\rbrack (t)} = {\frac{I_{e}ɛ\; c\; \tau_{f}}{h\; v_{e}} - {\frac{I_{e}ɛ\; c\; \tau_{f}}{h\; v_{e}}^{{- {({t - t_{1}})}}/\tau_{f}}}}$

At time t₂:

$\begin{matrix}{{\left\lbrack {S\; 1} \right\rbrack \left( t_{2} \right)} = {\frac{I_{e}ɛ\; c\; \tau_{f}}{h\; v_{e}} - {\frac{I_{e}ɛ\; c\; \tau_{f}}{h\; v_{e}}^{{- {({t_{2} - t_{1}})}}/\tau_{f}}}}} & (5)\end{matrix}$

For t≧t₂, the excited states decay exponentially and this is describedby:

[S1](t)=[S1](t ₂)e ^(−(t−t) ² ^()/τ) ^(f)   (6)

Substituting (5) into (6):

$\begin{matrix}{{\left\lbrack {S\; 1} \right\rbrack (t)} = {{\frac{I_{e}ɛ\; c\; \tau_{f}}{h\; v_{e}}\left\lbrack {1 - ^{{- {({t_{2} - t_{1}})}}/\tau_{f}}} \right\rbrack}^{{- {({t - t_{2}})}}/\tau_{f}}}} & (7)\end{matrix}$

The fluorescence intensity is given by the following equation:

$\begin{matrix}{{I_{f}(t)} = {{- \frac{{\left\lbrack {S\; 1} \right\rbrack}(t)}{x}}h\; v_{f}\eta \; l}} & (8)\end{matrix}$

where ν_(f) is the fluorescence frequency, η is the quantum yield and 1is the optical path length.

Now from (7):

$\begin{matrix}{\frac{{\left\lbrack {S\; 1} \right\rbrack}(t)}{t} = {{- {\frac{I_{e}ɛ\; c}{h\; v_{e}}\left\lbrack {1 - ^{{- {({t_{2} - t_{1}})}}/\tau_{f}}} \right\rbrack}}^{{- {({t - t_{2}})}}/\tau_{f}}}} & (9)\end{matrix}$

Substituting (9) into (8):

$\begin{matrix}{{{I_{f}(t)} = {I_{e}ɛ\; {cl}\; \eta {\frac{v_{f}}{v_{e}}\left\lbrack {1 - ^{{- {({t_{2} - t_{1}})}}/\tau_{f}}} \right\rbrack}^{{- {({t - t_{2}})}}/\tau_{f}}}}{\left. {{For}\mspace{14mu} \frac{t_{2} - t_{1}}{\tau_{f}}}\rightarrow\infty \right.,\left. {I_{f}(t)}\rightarrow{I_{e}ɛ\; {cl}\; \eta \frac{v_{f}}{v_{e}}^{{- {({t - t_{2}})}}/\tau_{f}}} \right.}} & (10)\end{matrix}$

Therefore, we can write the following approximate equation whichdescribes the fluorescence intensity decay after a sufficiently longexcitation pulse (t₂−t₁>>τ_(f)):

$\begin{matrix}{{I_{f}(t)} = {{I_{e}ɛ\; {cl}\; \eta \frac{v_{f}}{v_{e}}^{{- {({t - t_{2}})}}/\tau_{f}}\mspace{14mu} {for}\mspace{14mu} t} \geq t_{2}}} & (11)\end{matrix}$

In the previous section, we concluded that for t₂−t₁>>τ_(f),

${I_{f}(t)} = {{I_{e}ɛ\; {cl}\; \eta \frac{v_{f}}{v_{e}}^{{- {({t - t_{2}})}}/\tau_{f}}\mspace{14mu} {for}\mspace{14mu} t} \geq {t_{2}.}}$

From the above equation, we can derive the following:

$\begin{matrix}{{{\overset{\dddot{}}{n}}_{f}(t)} = {{\overset{\dddot{}}{n}}_{e}ɛ\; {cl}\; \eta \; ^{{- {({t - t_{2}})}}/\tau_{f}}}} & (12)\end{matrix}$

where

${{\overset{\dddot{}}{n}}_{f}(t)} = \frac{I_{f}(t)}{h\; v_{f}}$

is the number of fluorescent photons per unit time per unit area and

${\overset{\dddot{}}{n}}_{e} = \frac{I_{e}}{h\; v_{e}}$

is the number of excitation photons per unit time per unit area.

Consequently,

$\begin{matrix}{{{\overset{¨}{n}}_{f}(t)} = {\int_{t_{3}}^{\infty}{{{\overset{\dddot{}}{n}}_{f}(t)}{t}}}} & (13)\end{matrix}$

where {umlaut over (n)}_(f) is the number of fluorescent photons perunit area and t₃ is the instant of time at which the photodiode isturned on. Substituting (12) into (13):

$\begin{matrix}{{\overset{¨}{n}}_{f} = {\int_{t_{3}}^{\infty}{{\overset{\dddot{}}{n}}_{e}ɛ\; {cl}\; \eta \; ^{{- {({t - t_{2}})}}/\tau_{f}}{t}}}} & (14)\end{matrix}$

Now, the number of fluorescent photons that reach the photodiode perunit time per unit area,

(t), is given by the following:

$\begin{matrix}{{{\overset{\dddot{}}{n}}_{s}(t)} = {{{\overset{\dddot{}}{n}}_{f}(t)}\varphi_{0}}} & (15)\end{matrix}$

where φ₀ is the light gathering efficiency of the optical system.

Substituting (12) into (15) we find

$\begin{matrix}{{{\overset{\dddot{}}{n}}_{s}(t)} = {\varphi_{0}{\overset{\dddot{}}{n}}_{e}ɛ\; {cl}\; {\eta }^{{- {({t - t_{2}})}}/\tau_{f}}}} & (16)\end{matrix}$

Similarly, the number of fluorescence photons that reach the photodiodeper unit fluorescent area {umlaut over (n)}_(s), will be as follows:

${\overset{¨}{n}}_{s} = {\int_{t_{3}}^{\infty}{{{\overset{\dddot{}}{n}}_{s}(t)}{t}}}$

and substituting in (16) and integrating:

${\overset{¨}{n}}_{s} = {\varphi_{0}{\overset{\dddot{}}{n}}_{e}ɛ\; {cl}\; {\eta\tau}_{f}^{{- {({t_{3} - t_{2}})}}/\tau_{f}}}$

Therefore,

n _(s)=φ₀ {dot over (n)} _(e) εclητ _(f) e ^(−Δt/τ) ^(f)   (17)

The optimal value of t₃ is when the rate of electrons generated in thephotodiode 184 due to fluorescence photons becomes equal to the rate ofelectrons generated in the photodiode 184 by the excitation photons, asthe flux of the excitation photons decays much faster than that of thefluorescence photons.

The rate of sensor output electrons per unit fluorescent area due tofluorescence is:

${{\overset{\dddot{}}{e}}_{f}(t)} = {\varphi_{f}{{\overset{\dddot{}}{n}}_{s}(t)}}$

where φ_(f) is the quantum efficiency of the sensor at the fluorescencewavelength.

Substituting in (17) we have:

$\begin{matrix}{{{\overset{\dddot{}}{e}}_{f}(t)} = {\varphi_{f}\varphi_{0}{\overset{\dddot{}}{n}}_{e}ɛ\; {cl}\; \eta \; ^{{- {({t - t_{2}})}}/\tau_{f}}}} & (18)\end{matrix}$

Similarly, the rate of sensor output electrons per unit fluorescent areadue to the excitation photons is:

$\begin{matrix}{{{\overset{\dddot{}}{e}}_{e}(t)} = {\varphi_{e}{\overset{\dddot{}}{n}}_{e}^{{- {({t - t_{2}})}}/\tau_{e}}}} & (19)\end{matrix}$

where φ_(e) is the quantum efficiency of the sensor at the excitationwavelength, and τ_(e) is the time-constant corresponding to the “off”characteristics of the excitation LED. After time t₂, the LED's decayingphoton flux would increase the intensity of the fluorescence signal andextend its decay time, but we are assuming that this has a negligibleeffect on I_(f)(t), thus we are taking a conservative approach.

Now, as mentioned earlier, the optimal value of t₃ is when:

${{\overset{\dddot{}}{e}}_{f}\left( t_{3} \right)} = {{\overset{\dddot{}}{e}}_{e}\left( t_{3} \right)}$

Therefore, from (18) and (19) we have:

${\varphi_{f}\varphi_{0}{\overset{\dddot{}}{n}}_{e}ɛ\; {cl}\; \eta \; ^{{- {({t_{3} - t_{2}})}}/\tau_{f}}} = {\varphi_{e}{\overset{\dddot{}}{n}}_{e}^{{- {({t_{3} - t_{2}})}}/\tau_{e}}}$

and rearranging we find:

$\begin{matrix}{{t_{3} - t_{2}} = \frac{\ln \left( {ɛ\; {cl}\; \eta \frac{\varphi_{f}\varphi_{0}}{\varphi_{e}}} \right)}{\frac{1}{\tau_{f}} - \frac{1}{\tau_{e}}}} & (20)\end{matrix}$

From the previous two sections, we have the following two workingequations:

$\begin{matrix}{n_{s} = {\varphi_{0}{\overset{.}{n}}_{e}F\; \tau_{f}^{{- \Delta}\; {t/\tau_{f}}}}} & (21) \\{{\Delta \; t} = \frac{\ln \left( {F\frac{\varphi_{f}\varphi_{0}}{\varphi_{e}}} \right)}{\frac{1}{\tau_{f}} - \frac{1}{\tau_{e}}}} & (22)\end{matrix}$

where F=εclη and Δt=t₃−t₂. We also know that, in practice, t₂−t₁>>τ_(f).

The optimal time for fluorescence detection and the number offluorescence photons detected using the Philips LXK2-PR14-R00 LED andPulsar 650 dye are determined as follows. The optimum detection time isdetermined using equation (22):

Recalling the concentration of amplicon, and assuming that all ampliconshybridize, then the concentration of fluorescent fluorophores is:c=2.89(10)⁻⁶ mol/L

The height of the chamber is the optical path length 1=8(10)⁻⁶ m.

We have taken the fluorescence area to be equal to our photodiode area,yet our actual fluorescence area is substantially larger than ourphotodiode area; consequently we can approximately assume φ₀=0.5 for thelight gathering efficiency of our optical system. From the photodiodecharacteristics,

$\frac{\varphi_{f}}{\varphi_{e}} = 10$

is a very conservative value for the ratio of the photodiode quantumefficiency at the fluorescence wavelength to its quantum efficiency atthe excitation wavelength.

With a typical LED decay lifetime of τ_(e)=0.5 ns and using Pulsar 650specifications, Δt can be determined:

$\begin{matrix}{F = {{{\left\lbrack {1.48(10)^{6}} \right\rbrack \left\lbrack {2.89(10)^{- 6}} \right\rbrack}\left\lbrack {8(10)^{- 6}} \right\rbrack}(1)}} \\{= {3.42(10)^{- 5}}}\end{matrix}$ $\begin{matrix}{{\Delta \; t} = \frac{\ln \left( {\left\lbrack {3.42(10)^{- 5}} \right\rbrack (10)(0.5)} \right)}{\frac{1}{1(10)^{- 6}} - \frac{1}{0.5(10)^{- 9}}}} \\{= {4.34(10)^{- 9}\mspace{14mu} s}}\end{matrix}$

The number of photons detected is determined using equation (21). First,the number of excitation photons emitted per unit time {dot over(n)}_(e) is determined by examining the illumination geometry.

The Philips LXK2-PR14-R00 LED has a Lambertian radiation pattern,therefore:

$\begin{matrix}{{\overset{\dddot{}}{n}}_{l} = {{\overset{\dddot{}}{n}}_{l\; 0}{\cos (\theta)}}} & (23)\end{matrix}$

where

is the number of photons emitted per unit time per unit solid angle atan angle of θ off the LED's forward axial direction, and

is the valve of

in the forward axial direction.

The total number of photons emitted by the LED per unit time is:

$\quad\begin{matrix}\begin{matrix}{{\overset{.}{n}}_{l} = {\int_{\Omega}{{\overset{\dddot{}}{n}}_{l}\ {\Omega}}}} \\{= {\int_{\Omega}{{\overset{\dddot{}}{n}}_{l\; 0}\ {\cos (\theta)}{\Omega}}}}\end{matrix} & (24)\end{matrix}$

Now,

Δ Ω = 2π[1 − cos (θ + Δθ)] − 2π[1 − cos (θ)] $\begin{matrix}{{\Delta\Omega} = {2{\pi \left\lbrack {{\cos (\theta)} - {\cos \left( {\theta + {\Delta\theta}} \right)}} \right\rbrack}}} \\{= {{4\pi \; {\sin (\theta)}{\cos \left( \frac{\Delta \; \theta}{2} \right)}{\sin \left( \frac{\Delta \; \theta}{2} \right)}} + {4\pi \; {\cos (\theta)}{\sin^{2}\left( \frac{\Delta \; \theta}{2} \right)}}}}\end{matrix}$ Ω = 2πsin(θ)θ

Substituting this into (24):

$\quad\begin{matrix}{{\overset{.}{n}}_{l} = {\int_{0}^{\frac{\pi}{2}}{2\pi \; {\overset{\dddot{}}{n}}_{l\; 0}{\cos (\theta)}{\sin (\theta)}\ {\theta}}}} \\{= {\pi \; {\overset{\dddot{}}{n}}_{l\; 0}}}\end{matrix}$

Rearranging, we have:

$\begin{matrix}{{\overset{\dddot{}}{n}}_{l\; 0} = \frac{{\overset{.}{n}}_{l}}{\pi}} & (26)\end{matrix}$

The LED's output power is 0.515 W and ν_(e)=6.52(10)¹⁴ Hz, therefore:

$\quad\begin{matrix}\begin{matrix}{{\overset{.}{n}}_{l} = \frac{p_{l}}{h\; \nu_{e}}} \\{= \frac{0.515}{\left\lbrack {6.63(10)^{- 34}} \right\rbrack \left\lbrack {6.52(10)^{14}} \right\rbrack}} \\{= {1.19(10)^{18}\mspace{14mu} {photons}\text{/}s}}\end{matrix} & (27)\end{matrix}$

Substituting this value into (26) we have:

$\quad\begin{matrix}{{\overset{\dddot{}}{n}}_{l\; 0} = \frac{1.19(10)^{18}}{\pi}} \\{= {3.79(10)^{17}\mspace{14mu} {photons}\text{/}s\text{/}{sr}}}\end{matrix}$

Referring to FIG. 61, the optical centre 252 and the lens 254 of the LED26 are schematically shown. The photodiodes are 16 μm×16 μm, and for thephotodiode in the middle of the array, the solid angle (Ω) of the coneof light emitted from the LED 26 to the photodiode 184 is approximately:

$\quad\begin{matrix}{\Omega = {{area}\mspace{14mu} {of}\mspace{14mu} {{sensor}/r^{2}}}} \\{= \frac{\left\lbrack {16(10)^{- 6}} \right\rbrack \left\lbrack {16(10)^{- 6}} \right\rbrack}{\left\lbrack {2.825(10)^{- 3}} \right\rbrack^{2}}} \\{= {3.21(10)^{- 5}\mspace{14mu} {sr}}}\end{matrix}$

It will be appreciated that the central photodiode 184 of the photodiodearray 44 is used for the purpose of these calculations. A sensor locatedat the edge of the array would only receive 2% less photons upon ahybridization event for a Lambertian excitation source intensitydistribution.

The number of excitation photons emitted per unit time is:

$\quad\begin{matrix}\begin{matrix}{{\overset{.}{n}}_{e} = {{\overset{\dddot{}}{n}}_{l}\Omega}} \\{= {\left\lbrack {3.79(10)^{17}} \right\rbrack \left\lbrack {3.21(10)^{- 5}} \right\rbrack}} \\{= {1.22(10)^{13}\mspace{14mu} {photons}\text{/}s}}\end{matrix} & (28)\end{matrix}$

Now referring to equation (29):

$n_{s} = {\varphi_{0}{\overset{.}{n}}_{e}F\; \tau_{f}^{{- \Delta}\; {t/\tau_{f}}}}$$\begin{matrix}{n_{s} = {{{{(0.5)\left\lbrack {1.22(10)^{13}} \right\rbrack}\left\lbrack {3.42(10)^{- 5}} \right\rbrack}\left\lbrack {1(10)^{- 6}} \right\rbrack}^{{- 4.34}{{(10)}^{- 9}/1}{(10)}^{- 6}}}} \\{= {208\mspace{14mu} {photons}\mspace{14mu} {per}\mspace{14mu} {{sensor}.}}}\end{matrix}$

Therefore, using the Philips LXK2-PR14-R00 LED and Pulsar 650fluorophore, we can easily detect any hybridization events which resultsin this number of photons being emitted.

The SET LED illumination geometry is shown in FIG. 62. At I_(D)=20 mA,the LED has a minimum optical power output of p₁=240 μW centred atλ_(e)=340 nm (the absorption wavelength of the terbium chelate). Drivingthe LED at I_(D)=200 mA would increase the output power linearly top₁=2.4 mW. By placing the LED's optical centre 252, 17.5 mm away fromthe hybridization chamber array 110, we would approximately concentratethis output flux in a circular spot size which has a maximum diameter of2 mm.

The photon flux in the 2 mm-diameter spot at the hybridization awayplane is given by equation 27.

$\quad\begin{matrix}{{\overset{.}{n}}_{l} = \frac{p_{l}}{h\; \nu_{e}}} \\{= \frac{2.4(10)^{- 3}}{\left\lbrack {6.63(10)^{- 34}} \right\rbrack \left\lbrack {8.82(10)^{14}} \right\rbrack}} \\{= {4.10(10)^{15}\mspace{14mu} {photons}\text{/}s}}\end{matrix}$

Using equation 28, we have:

$\quad\begin{matrix}{{\overset{.}{n}}_{e} = {{\overset{\dddot{}}{n}}_{l}\Omega}} \\{= {4.10(10)15\frac{\left\lbrack {16(10)^{- 6}} \right\rbrack^{2}}{{\pi \left\lbrack {1(10)^{- 3}} \right\rbrack}^{2}}}} \\{= {3.34(10)^{11}\mspace{14mu} {photons}\text{/}s}}\end{matrix}$

Now, recalling equation 22 and using the Tb chelate properties listedpreviously,

$\quad\begin{matrix}{{\Delta \; t} = \frac{\ln \left\lbrack {\left( {6.94(10)^{- 5}} \right)(10)(0.5)} \right\rbrack}{\frac{1}{1(10)^{- 3}} - \frac{1}{0.5(10)^{- 9}}}} \\{= {3.98(10)^{- 9}\mspace{14mu} s}}\end{matrix}$

Now from equation 21:

$\quad\begin{matrix}{n_{s} = {{{{(0.5)\left\lbrack {3.34(10)^{11}} \right\rbrack}\left\lbrack {6.94(10)^{- 5}} \right\rbrack}\left\lbrack {1(10)^{- 3}} \right\rbrack}^{{- 3.98}{{(10)}^{- 9}/1}{(10)}^{- 3}}}} \\{= {11,600\mspace{14mu} {photons}\mspace{14mu} {per}\mspace{14mu} {{sensor}.}}}\end{matrix}$

The theoretical number of photons emitted by hybridization events usingthe SET LED and terbium chelate system are easily detectable and wellover the minimum of 30 photons required for reliable detection by thephotosensor as indicated above.

Maximum Spacing Between Probes and Photodiode

The on-chip detection of hybridization avoids the needs for detectionvia confocal microscopy (see Background of the Invention). Thisdeparture from traditional detection techniques is a significant factorin the time and cost savings associated with this system. Traditionaldetection requires imaging optics which necessarily uses lenses orcurved mirrors. By adopting non-imaging optics, the diagnostic systemavoids the need for a complex and bulky optical train. Positioning thephotodiode very close to the probes has the advantage of extremely highcollection efficiency: when the thickness of the material between theprobes and the photodiode is of the order of 1 micron, the angle ofcollection of emission light is up to 173°. This angle is calculated byconsidering light emitted from a probe at the centroid of the face ofthe hybridization chamber closest to the photodiode, which has a planaractive surface area parallel to that chamber face. The cone of emissionangles within which light is able to be absorbed by the photodiode isdefined as having the emitting probe at its vertex and the corner of thesensor on the perimeter of its planar face. For a 16 micron×16 micronsensor, the vertex angle of this cone is 170°; in the limiting casewhere the photodiode is expanded so that its area matches that of the 29micron×19.75 micron hybridization chamber, the vertex angle is 173°. Aseparation between the chamber face and the photodiode active surface of1 micron or less is readily achievable.

Employing a non-imaging optics scheme does require the photodiode 184 tobe very close to the hybridization chamber in order to collectsufficient photons of fluorescence emission. The maximum spacing betweenthe photodiode and probes is determined as follows with reference toFIG. 54.

Utilizing a terbium chelate fluorophore and a SET UVTOP335TO39BL LED, wecalculated 11600 photons reaching our 16 micron×16 micron photodiode 184from the respective hybridization chamber 180. In performing thiscalculation we assumed that the light-collecting region of ourhybridization chamber 180 has a base area which is the same as ourphotodiode active area 185, and half of the total number of thehybridization photons reaches the photodiode 184. That is, the lightgathering efficiency of the optical system is φ₀=0.5.

More accurately we can write φ₀=[(base area of the light-collectingregion of the hybridization chamber)/(photodiode area)][Ω/4π], whereΩ=solid angle subtended by the photodiode at a representative point onthe base of the hybridization chamber. For a right square pyramidgeometry:

Ω=4 arcsin(a ²/(4d ₀ ² +a ²)),

where d₀=distance between the chamber and the photodiode, and a is thephotodiode dimension.

Each hybridization chamber releases 23200 photons. The selectedphotodiode has a detection threshold of 17 photons; therefore, theminimum optical efficiency required is:

φ₀=17/23200=7.33×10⁻⁴

The base area of the light-collecting region of the hybridizationchamber 180 is 29 micron×19.75 micron.

Solving for d₀, we will get the maximum limiting distance between thebottom of our hybridization chamber and our photodiode 184 to be d₀=249microns. In this limit, the collection cone angle as defined above isonly 0.8°. It should be noted this analysis ignores the negligibleeffect of refraction.

LOC Variants

The LOC device 301 described and illustrated above in full is just oneof many possible LOC device designs. Variations of the LOC device thatuse different combinations of the various functional sections describedabove will now be described and/or shown as schematic flow-charts, fromsample inlet to detection, to illustrate some of the combinationspossible. The flow-charts have been divided, where appropriate, intosample input and preparation stage 288, extraction stage 290, incubationstage 291, amplification stage 292, pre-hybridization stage 293 anddetection stage 294. For all the LOC variants that are briefly describedor shown only in schematic form, the accompanying full layouts are notshown for reasons of clarity and succinctness. Also in the interests ofclarity, smaller functional units such as liquid sensors and temperaturesensors are not shown but it will be appreciated that these have beenincorporated into the appropriate locations in each of the following LOCdevice designs.

LOC Variant VII

FIGS. 82 to 96 show LOC variant VII 492. Features and structures thatcorrespond to equivalent features or structures shown in previousfigures are indicated by the same reference numeral.

As diagrammatically shown in FIG. 96, this variant extracts 290,incubates 291, amplifies 292 and detects 294 human DNA using parallelnucleic acid amplification. Four separate amplification sections 112.1to 112.4 are included to increase assay sensitivity and improvesignal-to-noise ratio of the detected fluorescence. The design also usesthe leukocyte dialysis section 328, lysis reagent 56, chemical lysissection 130, restriction enzymes, ligase and linkers 58 and incubationsection 114 as previously described. However, this LOC variant usesseveral of the fault tolerant valve arrays 309, 313, 462, 464, 466 and468 instead of single active valves at the output of the chemical lysissection 130, incubation section 114 and amplification sections 112.1 to112.4. The valve arrays are 2×2 arrays of the second type of thermalbend actuated valve 308.

Separate amplicons from each of the amplification sections 112.1 to112.4 feed into separate hybridization sections 110.1 to 110.4 wherefluorescence is detected by the photosensor 44.

FIG. 82 is a perspective of LOC variant VII 492 showing the vent holes122, the sample inlet 68 and the evaporator 190 in the upper seal layer82. Waste reservoirs 76 are unsealed such that excess waste can betransferred to a porous element 49 (see FIG. 1) within the test module.A series of bond-pads 104 extend along one edge and the humidity sensor232 is also exposed for sensing the humidity of the microenvironmentwithin the test module.

FIG. 83 is an exploded perspective of the cap 46 in isolation. Removingthe upper seal 82 reveals the reagent reservoirs (54, 56, 58, 188,60.1-60.4 and 62.1-62.4) beneath the vent holes 122. LOC variant VII 492has four amplification sections 112 to 112.4 and hence fouramplification mix reservoirs 60.1 to 60.4 and four polymerase reservoirs62.1 to 62.4, which provide the reagents for respective amplificationsections 112.1 to 112.4.

FIG. 84 is a perspective of the underside of the cap 46 showing theconfiguration of the cap channels 94 and the lower portions of thereagent reservoirs (60.1, 60.2, . . . 62.1, 62.2 . . . etc). FIG. 85superimposes the features of the cap 46 on the features of the CMOS+MSTdevice 48. FIG. 86 shows the features of the CMOS+MST device 48 inisolation. FIG. 87 shows the cap channels 94, reservoirs and valve arraycomponents in isolation. The waste channel 72 leads to the underside ofthe waste reservoirs 76 while the target channel 74 leads to thechemical lysis section 130 downstream of the lysis reagent reservoir 56.FIGS. 88, 89, 90, 91, 92, 93 and 94 are enlargements of Insets BA to BGrespectively.

The blood sample enters through the sample inlet 68. Capillary actiondraws the sample through the cap channel 94 to the anticoagulant surfacetension valve 118 (see FIG. 88). Anticoagulant from the reservoir 54combines with the blood sample which continues onto the leukocytedialysis section 328 (see FIG. 87). As best shown in FIG. 93, the targetchannel 74 and the waste cell channel 72 are connected by a series ofdialysis MST channels 204 through the MST layer 87. The target channel74 connects to the dialysis MST channels 204 through respective arraysof 7.5 micron holes 165. The dialysis MST channels 204 connect to thewaste channel 72 via dialysis uptakes 168. The dialysis MST channel 204at the upstream end 502 of the dialysis section has no downtake.Capillary initiation features 166 ensure that the sample flow does notpin at the array of 7.5 micron diameter apertures 165 but rather flowsthrough to the dialysis MST channels 204.

Referring back to FIGS. 85, 87 and 88, the sample flow, now with a muchhigher concentration of the target cells, flows to the lysis reagentsurface tension valve 128. Lysis reagent from the reservoir 56 combineswith the sample flow and enters into the chemical lysis section 130. Theflow stops at the 2×2 fault tolerant array 309 of thermal bend actuatedvalves of the second type 308. The CMOS circuitry 86 is programmed witha dwell time for diffusive mixing of the lysis reagent such that enoughof target cells are lysed. After sufficient time (less than 0.5 sec),the valves in the fault tolerant valve array 309 are activated (i.e.opened) and the flow continues into the downstream section of the mixingsection 131.

With the genetic material released, the restriction enzymes, ligase andlinkers from reservoir 58 are added via surface tension valve 132. Thesample flow continues through the remainder of the mixing section 131 tothe downtake 134 and into the heated microchannels of the incubationsection 114 (see FIG. 87). The sample flow fills the incubation section114 until it reaches the 2×2 fault tolerant valve array 313. Aftersufficient incubation time, the fault tolerant valve array 313activates.

The sample flows into the common supply channel 504 for theamplification sections. The common supply channel 504 feeds into theinlets 506, 508, 510 and 512 of the four separate amplification sections112.1 to 112.4 (see FIG. 89). Each of the amplification mix reservoirs60.1 to 60.4 has a surface tension valve 138 connecting to each of theamplification section inlets 506, 508, 510 and 512 respectively.Similarly, each of the polymerase reservoirs 62.1 to 62.4 have a surfacetension valve 140 connecting to each of the amplification section inlets506, 508, 510 and 512 respectively (see FIG. 87). The surface tensionvalves 138 and 140 retain the reagents in their respective reservoirs bypinning a meniscus. As the sample flows down each inlet, the meniscus isremoved and the reagents combine with the sample flow; firstly theamplification mix of primers, dNTPs and buffer, then the polymerase.

Each of the four separate amplification sections 112.1 to 112.4 fillswith the sample, amplification mix and polymerase. The respective flowthrough each of the amplification sections stops at the respectiveamplification exit fault tolerant valve arrays 462, 464, 466 and 468(see FIG. 90). The CMOS-controlled thermal cycling amplifies the geneticmaterial. Referring specifically to FIGS. 85, 91 and 95, theamplification exit valve arrays 462, 464, 466 and 468 activate such thatthe four amplicons flow into separate hybridization chamber arrays 110.1to 110.4 via separate inlets 494, 496, 498, 500. Each of the separatehybridization chamber arrays has its own end-point liquid sensor 178.Feedback from the respective end-point liquid sensors 178 triggers thehybridization heaters 182 and the LED driver 29 in the CMOS circuitrysignals the excitation LED 26 to illuminate. Hybridization with the FRETprobes 182 in any of the individual hybridization chambers 180 isdetected by the photodiodes 184 underlying those chambers (see FIG. 54).

CONCLUSION

The devices, systems and methods described here facilitate moleculardiagnostic tests at low cost with high speed and at the point-of-care.

The system and its components described above are purely illustrativeand the skilled worker in this field will readily recognize manyvariations and modifications which do not depart from the spirit andscope of the broad inventive concept.

1. A microfluidic device comprising: an inlet for receiving a liquidflowing through the microfluidic device; an outlet downstream of theinlet; and, a fault-tolerant multiple valve assembly having a pluralityof flow-paths extending from the inlet to the outlet and a plurality ofvalves positioned along each of the flow-paths respectively.
 2. Themicrofluidic device according to claim 1 wherein each of the flow-pathsis configured for capillary driven flow of the liquid towards theoutlet, and each of the valves are configured to arrest flow towards theoutlet until opened.
 3. The microfluidic device according to claim 2further comprising a sensor in each of the flow-paths respectively, thesensor being responsive to contact with the liquid.
 4. The microfluidicdevice according to claim 2 wherein the valves are bend actuated valves,each having a movable member configured for movement between a quiescentposition and an actuated position displaced from the quiescent positionand, an aperture at least partially defined by the movable member, theaperture configured to arrest the capillary driven flow by pinning ameniscus at the aperture wherein during use, displacement of the movablemember to the actuated position unpins the meniscus from the aperturesuch that the capillary driven flow towards the outlet resumes.
 5. Themicrofluidic device according to claim 4 wherein the bend actuated valvehas a resistive element for causing differential thermal expansion tomove the movable member.
 6. The microfluidic device according to claim 5wherein the movable member is a cantilevered structure with the nozzleat the free end and the resistive element between the nozzle and thefixed end.
 7. The microfluidic device according to claim 1 wherein thevalves are thermally actuated valves, each having an aperture foranchoring a meniscus to arrest the capillary drive flow, and a valveheater for heating the meniscus such that the meniscus unpins from theaperture such that the capillary driven flow towards the outlet resumes.8. The microfluidic device according to claim 7 wherein the valve heaterextends about the periphery of the aperture.
 9. The microfluidic deviceaccording to claim 3 wherein each of the flow-paths has an upstreamvalve and a downstream valve, the sensor being positioned between theupstream valve and the downstream valves.
 10. The microfluidic deviceaccording to claim 9 further comprising two of the flow-paths.
 11. Themicrofluidic device according to claim 9 further comprising CMOScircuitry connected to the sensors for operative control of the valvesand detecting faults including failure of either of the upstream valvesto arrest the liquid flow.
 12. The microfluidic device according toclaim 1 further comprising a polymerase chain reaction (PCR) section foramplifying nucleic acid sequences by thermally cycling the nucleic acidsequences and a PCR mix of reagents through a denaturation temperature,an annealing temperature and a primer extension temperature; wherein,the fault-tolerant multiple valve assembly retains the nucleic acidsequences and a PCR mix of reagents in the PCR section during thethermal cycling.
 13. The microfluidic device according to claim 12further comprising an array of probes for hybridization with targetnucleic acid sequences to form probe-target hybrids; the array of probesbeing downstream of the fault-tolerant multiple valve assembly such thatopening the fault-tolerant multiple valve assembly allows amplicon fromthe PCR section to contact the probes.
 14. The microfluidic deviceaccording to claim 13 wherein the PCR section is configured to producesufficient amplicon for hybridization with more than 1000 of the probesin less than 10 minutes of thermal cycling.
 15. The microfluidic deviceaccording to claim 14 wherein the PCR section has a thermal cycle timebetween 0.45 seconds and 1.5 seconds.
 16. The microfluidic deviceaccording to claim 9 further comprising a temperature sensor and whereinthe PCR section has at least one heater for thermally cycling thenucleic acid sequences and the PCR mix, the temperature sensor and theat least one heater being connected to the CMOS circuitry for feedbackcontrol of the at least one heater.
 17. The microfluidic deviceaccording to claim 9 wherein the CMOS circuitry opens the fault-tolerantvalve assembly after a predetermined number of thermal cycles.
 18. Themicrofluidic device according to claim 12 wherein the PCR section has aplurality of elongate PCR chambers having a longitudinal extent muchgreater than their lateral dimensions, and a plurality of heaters, eachof the heaters being elongate and parallel with the longitudinal extentof the PCR chambers.
 19. The microfluidic device according to claim 18wherein the plurality of elongate heaters are independently operable.20. The microfluidic device according to claim 9 wherein the probes arefluorescent resonant energy transfer (FRET) probes and the CMOScircuitry further comprises an array of photodiodes for detectinghybridization of probes within the array of probes.